CN105821029A - Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof - Google Patents
Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN105821029A CN105821029A CN201510001075.6A CN201510001075A CN105821029A CN 105821029 A CN105821029 A CN 105821029A CN 201510001075 A CN201510001075 A CN 201510001075A CN 105821029 A CN105821029 A CN 105821029A
- Authority
- CN
- China
- Prior art keywords
- cell
- galactosyl transferase
- arbitrary
- recombinant vector
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and its preparation method. According to the heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and its preparation method, a method for cell fusion comprises a step of fusing cancer cell which contains Endoglin and alpha1,3 galactosyl transferase related gene and is used for expression of Endoglin and alpha1,3 galactosyl transferase and dendritic cell to obtain fusion cell. The alpha1,3 galactosyl transferase gene encodes protein with its amino acid sequence being SEQ ID No.3. The Endoglin is protein with its amino acid sequence being SEQ ID No.1. The alpha1,3 galactosyl transferase related gene is formed by connecting the alpha1,3 galactosyl transferase gene and GTTI. Nucleotide sequence of GTTI is from the 1st nucleotide to the 548th nucleotide in the SEQ ID No.5 of the sequence table.
Description
Technical field
The present invention relates to the cancerous cell of heterolgous fusion genetic modification in biomedical sector/dendritic cell and merge tumor vaccine and preparation method thereof.
Background technology
The immunization therapy of malignant tumor has become as a kind of new Therapeutic Method outside operation, radiation and chemotherapy.Wherein tumor-specific cytotoxicity T lymphocyte (cytotoxicTlymphocyte, CTL) therapy of adopting is always the research emphasis of immunotherapy of tumors.Tumor cell and dendritic cell (dendriticcells, DC) merge the effective ways as a kind of CTL of induction and are widely accepted.But the therapeutic effect of fusion bacterin is the most undesirable, new method is needed to strengthen effectiveness and the targeting of fused cell induction CTL.
Endoglin is (also known as CD105, differentiation group 105) it is a kind of homodimer transmembrane glycoprotein tumor vascular labelling molecule, be molecular weight be 180kDa, it is transforming growth factor-β (transforminggrowthfactor, TGF-β) part of receptor, the reaction to TGF-β of the controllable cell, promotes endothelial cell proliferation by the biological effect of suppression TGF-β.
At α 1,3 galactosyl transferases [alpha (1,3) Galactosyltransferase, alpha (1,3) GT] catalysis under, the glycosyl on uridine diphosphate galactose transfer on the N-Acetyl-D-glucosamine residue in glycolipid and glycoprotein chains formed α-Gal antigen.α tilactase antigen (α-Gal or Gal α 1-3Gal β 1-4GlcNAc-R) is a kind of special carbohydrate structure, end α-galactose residue is natural antibody specific recognition sites, must be with alpha form 1,3 connections between galactose and galactose.
Summary of the invention
The technical problem to be solved is how to treat tumor.
For solving above-mentioned technical problem, present invention firstly provides the method preparing fused cell.
The method preparing fused cell provided by the present invention, for S1 or S2:
S1, described fused cell are fused cell 1；The described method preparing fused cell 1 includes carrying out tumor cell 1 and dendritic cell merging the step obtaining fused cell 1；
Described tumor cell 1 is M or N:
M, described tumor cell 1 are for expressing Endoglin and α 1, the reconstitution cell of 3 galactosyl transferases；
N, described tumor cell 1 are containing gene and α 1, the reconstitution cell of 3 galactosyl transferase related genes encoding described Endoglin；
Described Endoglin is following A 1) or protein A2):
A1) aminoacid sequence is the protein of SEQIDNo.1；
A2) in the aminoacid sequence shown in SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) derivative protein；
Described α 1,3 galactosyl transferase is following B1) or protein B2):
B1) aminoacid sequence is the protein of SEQIDNo.3；
B2) in the aminoacid sequence shown in SEQIDNo.3 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have described α 1,3 galactosyl transferase function by B1) derivative protein；
Described α 1,3 galactosyl transferase related gene is formed by connecting by α 1, the DNA molecular of 3 galactosyl transferase genes and entitled GTTI；
α 1,3 galactosyl transferase described in described α 1,3 galactosyl transferase gene code；
The nucleotides sequence of described GTTI is classified as the 1st-the 548 nucleotide of SEQ ID No .5；
S2, described fused cell are fused cell 2；The described method preparing fused cell 2 includes carrying out tumor cell 2 and dendritic cell merging the step obtaining fused cell 2；
Described tumor cell 2 is that 3 galactosyl transferase genes also express described Endoglin and the reconstitution cell of described α 1,3 galactosyl transferase containing gene and the α 1 encoding described Endoglin.
Wherein, SEQIDNo.5 is made up of 4063 nucleotide, the nucleotide sequence that the 1st-the 548 nucleotide is described GTTI of SEQIDNo.5；The coded sequence that 581st-the 1630 nucleotide is described α 1,3 galactosyl transferase of SEQIDNo.5, encoding amino acid sequence is the protein of SEQ ID No .3；The 1st-the 1630 nucleotide of SEQIDNo.5 is α 1, the nucleotide sequence of 3 galactosyl transferase related genes；The coded sequence that 1640th-the 3322 nucleotide is described Endoglin of SEQIDNo.5, encoding amino acid sequence is the protein of SEQ ID No .1；The 3344th-the 4063 nucleotide of SEQIDNo.5 is the coded sequence of EGFP.
Above-mentioned A2) in protein can synthetic, it is possible to first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned A2) in the encoding gene of protein can be by the codon by lacking one or several amino acid residue in the DNA sequence shown in the 1640th of SEQIDNo.5 the-the 3322 nucleotide, and/or carry out the missense mutation of one or several base pair, and/or connect the coded sequence of the fluorescin shown in the 3344th-the 4063 nucleotide of SEQIDNo.5 at its 5 ' end and/or 3 ' ends and obtain.
Above-mentioned B2) in protein can synthetic, it is possible to first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned B2) in the encoding gene of protein can be by the codon by lacking one or several amino acid residue in the DNA sequence shown in the 581st of SEQIDNo.5 the-the 1630 nucleotide, and/or carry out the missense mutation of one or several base pair, and/or connect the coded sequence of the fluorescin shown in the 3344th-the 4063 nucleotide of SEQIDNo.5 at its 5 ' end and/or 3 ' ends and obtain.
In the above-mentioned method preparing fused cell, described tumor cell 1 is that encoding gene and described α 1, the 3 galactosyl transferase related gene of described Endoglin are imported the reconstitution cell obtained in receptor tumor cells；Described tumor cell 2 is the reconstitution cell that will obtain in receptor tumor cells described in the encoding gene of described Endoglin and described α 1,3 galactosyl transferase channel genes.
In the above-mentioned method preparing fused cell, the encoding gene of described Endoglin is following A11) or A21) or A31) shown in nucleic acid molecules:
A11) the 1640th-the 3322 DNA molecular shown in nucleotide of SEQ ID No .5；
A21) nucleotide sequence and A11) limited has 75% or more than 75% homogeneity, and encodes cDNA molecule or the genomic DNA molecule of described Endoglin；
A31) under strict conditions with A11) nucleotide sequence hybridization that limits, and encode cDNA molecule or the genomic DNA molecule of described Endoglin；
Described α 1,3 galactosyl transferase gene is following B11) or B21) or B31) shown in nucleic acid molecules:
B11) the 581st-the 1630 DNA molecular shown in nucleotide of SEQ ID No .5；
B21) nucleotide sequence and B11) limited has 75% or more than 75% homogeneity, and encodes cDNA molecule or the genomic DNA molecule of described α 1,3 galactosyl transferase；
B31) under strict conditions with B11) nucleotide sequence hybridization that limits, and encode cDNA molecule or the genomic DNA molecule of described α 1,3 galactosyl transferase.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes that the 1640th-the 3322 nucleotide sequence of the SEQIDNo.5 with the present invention or the 581st-the 1630 nucleotide sequence of SEQIDNo.5 have 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to the homogeneity evaluating between correlated series.
In the above-mentioned method preparing fused cell, described stringent condition is at 2 × SSC, in the solution of 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, and each 5min, again in 0.5 × SSC, in the solution of 0.1%SDS, hybridizes at 68 DEG C and washes film 2 times, each 15min.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%.
In the above-mentioned method preparing fused cell, the C-terminal of described Endoglin or described α 1,3 galactosyl transferase or N-terminal can merge His, Flag, GST, MBP, His-MBP, HA, eGFP, eCFP, eYFP, Myc, His-Myc, His-AviTag, Sumo, His-Sumo, SNAP-Tag or HaloTag label.
In the above-mentioned method preparing fused cell, described α 1,5 ' ends of 3 galactosyl transferase related genes and/or 3 ' ends can connect the coded sequence of conventional label, it is possible at 5 ' ends and/or the coded sequence of the 3 ' conventional labels of end connection of described α 1,3 galactosyl transferase gene.
In the above-mentioned method preparing fused cell, described conventional label can be His, Flag, GST, MBP, His-MBP, HA, EGFP, eCFP, eYFP, Myc, His-Myc, His-AviTag, Sumo, His-Sumo, SNAP-Tag or HaloTag label.
In one embodiment of the invention, described α 1,3 ' ends of 3 galactosyl transferase related genes connect the encoding gene of described Endoglin, 3 ' ends of the encoding gene of described Endoglin connect the encoding gene of described EGFP, and the nucleotide sequence after the connection of these three gene is as shown in SEQ ID No .5.
In the above-mentioned method preparing fused cell, the nucleotides sequence of described α 1,3 galactosyl transferase related gene is classified as the 1st-the 1630 nucleotide of SEQ ID No .5；
Described tumor cell 1 expresses described Endoglin and containing described α 1,3 galactosyl transferase related gene.
In the above-mentioned method preparing fused cell, described tumor cell 1 can be for importing the reconstitution cell obtained in described receptor tumor cells by the DNA molecular shown in the 1st of SEQIDNo.5 the-the 3322 nucleotide；
Described tumor cell 2 can be for importing the reconstitution cell obtained in described receptor tumor cells by the DNA molecular shown in the 581st of SEQIDNo.5 the-the 3322 nucleotide.
In one embodiment of the invention, described tumor cell 1 is that the DNA molecular shown in SEQIDNo.5 is imported the reconstitution cell obtained in receptor tumor cells.
In the above-mentioned method preparing fused cell, described tumor cell can be hepatoma carcinoma cell, concretely HepG2.
In the above-mentioned method preparing fused cell, described dendritic cell can be the dendritic cell of preparation, the peripheral blood of described peripheral blood concretely people from peripheral blood.
For solving above-mentioned technical problem, present invention also offers fused cell.
Fused cell provided by the present invention is the fused cell that the above-mentioned method preparing fused cell obtains.
For solving above-mentioned technical problem, present invention also offers treatment and/or prophylaxis of tumours medicine.
Treatment provided by the present invention and/or the active component of prophylaxis of tumours medicine, for following H1 and/or H2:
H1, described fused cell；
The T lymphocyte of the secretion of gamma-IFN that fused cell inducer T lymphocyte described in H2 obtains.
In above-mentioned treatment and/or prophylaxis of tumours medicine, described treatment and/or prophylaxis of tumours medicine can be treatment and/or the medicine that prevents liver cancer.
For solving above-mentioned technical problem, present invention also offers any one product in following X1-X9:
X1, fusion protein, for following X1a) or protein X1b):
X1a) by the protein of the 581st of SEQIDNo.5 the-the 3322 nucleotide coding；
X1b) at X1a) protein aminoacid sequence in through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by X1a) derivative protein；
The encoding gene of fusion protein described in X2, X1, for the 1st-the 3322 nucleotide of SEQIDNo.5；
The encoding gene of fusion protein described in X3, X1, for the 581st-the 3322 nucleotide of SEQIDNo.5；
X4, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is made up of described α 1,3 galactosyl transferase related gene and described Endoglin；
X5, for prepare treatment and/or the product of prophylaxis of tumours medicine, its active component by described α 1,3 galactosyl transferase related gene and described Endoglin encoding gene form；
X6, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is made up of described α 1,3 galactosyl transferase gene and described Endoglin；
X7, for prepare treatment and/or the product of prophylaxis of tumours medicine, its active component by described α 1,3 galactosyl transferase gene and described Endoglin encoding gene form；
X8, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is made up of described α 1,3 galactosyl transferase and described Endoglin；
X9, for prepare treatment and/or the product of prophylaxis of tumours medicine, its active component by described α 1,3 galactosyl transferase and described Endoglin encoding gene form.
In the said goods, the 1st-the 3322 DNA molecular shown in nucleotide of the concretely SEQIDNo.5 of the product described in X5.
In the said goods, the 581st-the 3322 DNA molecular shown in nucleotide of the concretely SEQIDNo.5 of the product described in X7.
In the said goods, described treatment and/or prophylaxis of tumours medicine can be treatment and/or the medicine that prevents liver cancer.
For solving above-mentioned technical problem, present invention also offers the application of following N1 or N2 or N3 or N4 or N5:
The application in preparation treatment and/or prophylaxis of tumours medicine of N1, described fused cell or the application in the T lymphocyte of preparation secretion of gamma-IFN；
The application in preparation treatment and/or prophylaxis of tumours medicine of N2, described product；
The application treated and/or in prophylaxis of tumours medicine prepared by biomaterial and the biomaterial relevant with described α 1,3 galactosyl transferase related gene that N3 is relevant to described Endoglin；
The described biomaterial relevant to Endoglin is following E1) to E20) in any one:
E1) nucleic acid molecules of described Endoglin is encoded；
E2) containing E1) expression cassette of described nucleic acid molecules；
E3) containing E1) recombinant vector of described nucleic acid molecules；
E4) containing E2) recombinant vector of described expression cassette；
E5) containing E1) recombinant microorganism of described nucleic acid molecules；
E6) containing E2) recombinant microorganism of described expression cassette；
E7) containing E3) recombinant microorganism of described recombinant vector；
E8) containing E4) recombinant microorganism of described recombinant vector；
E9) containing E1) the transgenetic animal cell system of described nucleic acid molecules；
E10) containing E2) the transgenetic animal cell system of described expression cassette；
E11) containing E3) the transgenetic animal cell system of described recombinant vector；
E12) containing E4) the transgenetic animal cell system of described recombinant vector；
E13) containing E1) the transgenic animal tissue of described nucleic acid molecules；
E14) containing E2) the transgenic animal tissue of described expression cassette；
E15) containing E3) the transgenic animal tissue of described recombinant vector；
E16) containing E4) the transgenic animal tissue of described recombinant vector；
E17) containing E1) transgenic animal organ of described nucleic acid molecules；
E18) containing E2) transgenic animal organ of described expression cassette；
E19) containing E3) transgenic animal organ of described recombinant vector；
E20) containing E4) transgenic animal organ of described recombinant vector；
Described biomaterial relevant with α 1,3 galactosyl transferase related gene is following F1) to F19) in any one:
F1) expression cassette containing described α 1,3 galactosyl transferase related gene；
F2) recombinant vector containing described α 1,3 galactosyl transferase related gene；
F3) containing F1) recombinant vector of described expression cassette；
F4) recombinant microorganism containing described α 1,3 galactosyl transferase related gene；
F5) containing F1) recombinant microorganism of described expression cassette；
F6) containing F2) recombinant microorganism of described recombinant vector；
F7) containing F3) recombinant microorganism of described recombinant vector；
F8) the transgenetic animal cell system containing described α 1,3 galactosyl transferase related gene；
F9) containing F1) the transgenetic animal cell system of described expression cassette；
F10) containing F2) the transgenetic animal cell system of described recombinant vector；
F11) containing F3) the transgenetic animal cell system of described recombinant vector；
F12) the transgenic animal tissue containing described α 1,3 galactosyl transferase related gene；
F13) containing F1) the transgenic animal tissue of described expression cassette；
F14) containing F2) the transgenic animal tissue of described recombinant vector；
F15) containing F3) the transgenic animal tissue of described recombinant vector；
F16) transgenic animal organ containing described α 1,3 galactosyl transferase related gene；
F17) containing F1) transgenic animal organ of described expression cassette；
F18) containing F2) transgenic animal organ of described recombinant vector；
F19) containing F3) transgenic animal organ of described recombinant vector；
Biomaterial that N4 is relevant to described Endoglin and with the application in preparation treatment and/or prophylaxis of tumours medicine of the biomaterial of described α 1,3 galactosyl transferase gene-correlation；
Described is following G1 with described α 1,3 galactosyl transferase gene-correlation biomaterial) to G19) in any one:
G1) expression cassette containing described α 1,3 galactosyl transferase gene；
G2) recombinant vector containing described α 1,3 galactosyl transferase gene；
G3) containing G1) recombinant vector of described expression cassette；
G4) recombinant microorganism containing described α 1,3 galactosyl transferase gene；
G5) containing G1) recombinant microorganism of described expression cassette；
G6) containing G2) recombinant microorganism of described recombinant vector；
G7) containing G3) recombinant microorganism of described recombinant vector；
G8) the transgenetic animal cell system containing described α 1,3 galactosyl transferase gene；
G9) containing G1) the transgenetic animal cell system of described expression cassette；
G10) containing G2) the transgenetic animal cell system of described recombinant vector；
G11) containing G3) the transgenetic animal cell system of described recombinant vector；
G12) the transgenic animal tissue containing described α 1,3 galactosyl transferase gene；
G13) containing G1) the transgenic animal tissue of described expression cassette；
G14) containing G2) the transgenic animal tissue of described recombinant vector；
G15) containing G3) the transgenic animal tissue of described recombinant vector；
G16) transgenic animal organ containing described α 1,3 galactosyl transferase gene；
G17) containing G1) transgenic animal organ of described expression cassette；
G18) containing G2) transgenic animal organ of described recombinant vector；
G19) containing G3) transgenic animal organ of described recombinant vector；
The application in preparation treatment and/or prophylaxis of tumours medicine of the T lymphocyte of the secretion of gamma-IFN that N5, described fused cell inducer T lymphocyte obtain.
In above-mentioned application, the expression cassette of the nucleic acid molecules containing coding Endoglin described in E2), refer to express the DNA of Endoglin in host cell, this DNA not only can include starting the promoter that the encoding gene of Endoglin is transcribed, and may also include and terminates the terminator that Endoglin encoding gene is transcribed.Further, described expression cassette may also include enhancer sequence.
In above-mentioned application, F1) described in containing α 1, the expression cassette of 3 galactosyl transferase related genes, not only can include starting α 1, the promoter that 3 galactosyl transferase related genes are transcribed, may also include and terminate the terminator that α 1,3 galactosyl transferase related gene is transcribed.Further, described expression cassette may also include enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of described Endoglin encoding gene expression cassette.
Available existing expression vector establishment contains the recombinant vector of described α 1,3 galactosyl transferase related gene expression box.
In above-mentioned application, described carrier can be plasmid, glutinous grain, phage or viral vector.
In above-mentioned application, described microorganism can be yeast, antibacterial, algae or fungus.
In above-mentioned application, described transgenetic animal cell system, described transgenic animal tissue and described transgenic animal organ the most do not include propagating materials.
In an embodiment of the invention, the encoding gene of described Endoglin is imported in hepatocellular carcinoma H22 by the recombinant vector of the expression cassette of the encoding gene containing described Endoglin.E3) or E4) described recombinant vector is the recombinant vector that the fragment replacing on pLVX-Puro carrier between BamH I and Xba I recognition site with the DNA molecular shown in SEQ ID No .2 obtains.
Wherein, the 1st-the 1683 nucleotide of SEQIDNo.2, identical with the 1640th of SEQIDNo.5 the-the 3322 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.1；The 1705th-the 2424 nucleotide coding EGFP of SEQIDNo.2 is identical with the 3344th of SEQIDNo.5 the-the 4063 nucleotide.
In an embodiment of the invention, described α 1,3 galactosyl transferase related gene is imported in hepatocellular carcinoma H22 by the recombinant vector of the expression cassette containing described α 1,3 galactosyl transferase related gene.F2) or F3) described recombinant vector is the recombinant vector that the fragment replacing on pLVX-Puro carrier between BamH I and Xba I recognition site with the DNA molecular shown in SEQ ID No .4 obtains.
Wherein, the 1st-the 1630 nucleotide of SEQIDNo.4 is α 1, the nucleotide sequence of 3 galactosyl transferase related genes, and identical with the 1st of SEQIDNo.5 the-the 1630 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.3；The 581st-the 1630 nucleotide of SEQIDNo.4 is α 1,3 galactosyl transferase gene coded sequences, and identical with the 581st of SEQIDNo.5 the-the 1630 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.3；The DNA molecular that 1st-the 548 nucleotide is entitled GTTI of SEQIDNo.4 is identical with the 1st of SEQIDNo.5 the-the 548 nucleotide；The 1646th-the 2365 nucleotide coding EGFP of SEQIDNo.4 is identical with the 3344th of SEQIDNo.5 the-the 4063 nucleotide.
In an embodiment of the invention, described transgenetic animal cell is that the DNA molecular shown in SEQIDNo.5 is imported the reconstitution cell obtained in hepatocellular carcinoma H22.
In above-mentioned application, described treatment and/or prophylaxis of tumours medicine can be treatment and/or the medicine that prevents liver cancer.
For solve above-mentioned technical problem, present invention also offers following P1)-P7) in any one treatment and/or prophylaxis of tumours medicine:
P1) treatment and/or prophylaxis of tumours medicine prepared by described fused cell, are utilized；
Treatment prepared by the biomaterial P2), utilizing the described biomaterial relevant to Endoglin and described and α 1,3 galactosyl transferase related gene to be correlated with and/or prophylaxis of tumours medicine；
P3) the described biomaterial relevant to Endoglin and the described treatment prepared with α 1,3 galactosyl transferase gene-correlation biomaterial and/or prophylaxis of tumours medicine, are utilized；
P4) treatment and/or prophylaxis of tumours medicine prepared by described product, are utilized；
P5) treatment and/or prophylaxis of tumours medicine prepared by described tumor cell 1, are utilized；
P6) treatment and/or prophylaxis of tumours medicine prepared by described tumor cell 2, are utilized；
P7) treatment and/or prophylaxis of tumours medicine prepared by the T lymphocyte of the secretion of gamma-IFN that described fused cell inducer T lymphocyte obtains, are utilized.
In above-mentioned treatment and/or prophylaxis of tumours medicine, described treatment and/or prophylaxis of tumours medicine can be treatment and/or the medicine that prevents liver cancer.
In the present invention, T lymphocyte can be the T lymphocyte deriving from human peripheral.
nullExperiment proves，The T lymphocyte of the secretion of gamma-IFN that the fused cell DC/HepG2 (GT-Eng+) of the present invention induces can significantly delay tumor growth: the volume of the tumor after the T lymphocyte treatment of the secretion of gamma-IFN induced through DC/HepG2 (GT-Eng+) is respectively PBS、DC/HepG2、DC/HepG2(Eng+)、DC/HepG2(GT+)、DC/HepG2(pLVX-Puro)、DC、HepG2、HepG2(Eng+)、HepG2 (GT+) and 0.079 times of HepG2 (pLVX-Puro)、0.111 times、0.276 times、0.274 times、0.131 times、0.107 times、0.070 times、0.068 times、0.072 times、0.074 times.nullThe T lymphocyte of the secretion of gamma-IFN that the fused cell DC/HepG2 (GT-Eng+) of the present invention induces can be obviously prolonged lotus people's Liver Cancer Bearing Nude Mice life span: with PBS、DC/HepG2(GT+)、DC/HepG2(Eng+)、DC/HepG2(pLVX-Puro)、DC/HepG2、DC、HepG2、HepG2(Eng+)、HepG2(GT+)、HepG2 (GT-Eng+) and HepG2 (pLVX-Puro) compares，Survival rate at lotus people's Liver Cancer Bearing Nude Mice is respectively 80%、60%、40%、When 20% and 0，The lotus people's Liver Cancer Bearing Nude Mice of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) the induces time away from the 1st treatment all extends.It is demonstrated experimentally that the T lymphocyte of secretion of gamma-IFN that the DC/HepG2 of the application (GT-Eng+) induces can substantially suppress tumor growth, extend lotus people's Liver Cancer Bearing Nude Mice life span.
Accompanying drawing explanation
Fig. 1 is the order of connection of GTTI, alpha (1,3) GT, Endoglin and EGFP in SEQIDNo.5.
Fig. 2 is the expression of Endoglin albumen in Westernblot method detection HepG2 (GT-Eng+).
Fig. 3 is the positive rate that immuno-fluorescence assay expresses the HepG2 (GT-Eng+) of alpha (1,3) GT.
Fig. 4 is the Phenotypic examination of ripe DC.
Fig. 5 is the T lymphocyte treatment tumor presence of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces.Wherein, A is the situation of change of gross tumor volume, and B is the survival condition of nude mice.Wherein, DC/HepG2 (Gal-Eng+) represents DC/HepG2 (GT-Eng+).
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for illustrating the present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
PLVX-Puro carrier in following embodiment is Nanjing Jin Sirui Products, and article No. is RP20513.
HepG2 cell in following embodiment is bought from ATCC cell bank, and article No. is HB-8065TM。
Inbred line Female nude mice (BALB/cNudeMice) in following embodiment is bought from Shanghai Bang Yao Bioisystech Co., Ltd.
RhGM-CSF (RecombinanthumanGranulocyte/Macrophagecolony-stimulating factor, recombined human granulocyte macrophage colony simulating factor) in following embodiment is R&DSystems Products, and article No. is 215-GM-010；RhIL-4 (Recombinanthumaninterleukin-4, recombinant human interleukin--4) is R&DSystems Products, and article No. is 204-IL-010；RhIL-2 (Recombinanthumaninterleukin-2, recombinant human interleukin--2) is R&DSystems Products, and article No. is 202-IL-010；RhTNF-α (Recombinanthumantumornecrosisfactor-α, recombination human tumor necrosis factor-alpha) is R&DSystems Products, and article No. is 210-TA-005.
T lymphocyte in following embodiment is the T lymphocyte deriving from human peripheral.
ISOLECTINGS-IB4 in following embodiment is Invitrogen Products, and article No. is I32450, and this ISOLECTINGS-IB4 combines Alexa647。
Embodiment 1, the preparation of fused cell
The preparation method of fused cell, including S1) and S2)；
S1) encoding gene and α 1, the 3 galactosyl transferase related gene of transform growth factor-beta receptor associated protein are imported in tumor cell and obtain reconstitution cell；
S2) described reconstitution cell is obtained described fused cell with dendritic cell fusion.
Concrete grammar is as follows:
One, the preparation of reconstitution cell
1, the preparation of reconstitution cell
Fragment between BamH on pLVX-Puro carrier I and Xba I recognition site is replaced with the DNA molecular shown in SEQ ID No .5, keep other sequences constant, obtain recombinant vector pLVX-Puro/GT-Eng, this recombinant vector pLVX-Puro/GT-Eng expresses the Endoglin albumen shown in SEQIDNo.1 and the α 1 shown in SEQIDNo.3,3 galactosyl transferases (alpha (1,3) Galactosyltransferase, alpha (1,3) GT).This recombinant vector pLVX-Puro/GT-Eng is imported in HepG2 cell, obtain containing α 1,3 galactosyl transferases (alpha (1,3) Galactosyltransferase, alpha (1,3) GT) related gene coded sequence, Endoglin coded sequence and the reconstitution cell of EGFP coded sequence, by named for this reconstitution cell HepG2 (GT-Eng+).
Wherein, SEQIDNo.5 is made up of 4063 nucleotide, the nucleotide sequence that the 1st-the 548 nucleotide is described GTTI of SEQIDNo.5；The coded sequence that 581st-the 1630 nucleotide is described α 1,3 galactosyl transferase of SEQIDNo.5, encoding amino acid sequence is the protein of SEQ ID No .3；The 1st-the 1630 nucleotide of SEQIDNo.5 is α 1, the nucleotide sequence of 3 galactosyl transferase related genes；The coded sequence that 1640th-the 3322 nucleotide is described Endoglin of SEQIDNo.5, encoding amino acid sequence is the protein of SEQ ID No .1；The coded sequence (Fig. 1) that 3344th-the 4063 nucleotide is EGFP of SEQIDNo.5.
Fragment between BamH on pLVX-Puro carrier I and Xba I recognition site is replaced with the DNA molecular shown in SEQ ID No .2, keep other sequences constant, obtaining recombinant vector pLVX-Puro/Eng, this recombinant vector pLVX-Puro/Eng expresses the Endoglin albumen shown in SEQIDNo.1.This recombinant vector pLVX-Puro/Eng is imported in HepG2 cell, obtains containing Endoglin coded sequence and the reconstitution cell of EGFP coded sequence, by named for this reconstitution cell HepG2 (Eng+).
Wherein, the 1st-the 1683 nucleotide of SEQIDNo.2, identical with the 1640th of SEQIDNo.5 the-the 3322 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.1；The 1705th-the 2424 nucleotide coding EGFP of SEQIDNo.2 is identical with the 3344th of SEQIDNo.5 the-the 4063 nucleotide.
Fragment between BamH on pLVX-Puro carrier I and Xba I recognition site is replaced with the DNA molecular shown in SEQ ID No .4, keep other sequences constant, obtain recombinant vector pLVX-Puro/GT, this recombinant vector pLVX-Puro/GT expresses the α 1 shown in SEQIDNo.3,3 galactosyl transferases (alpha (1,3) Galactosyltransferase, alpha (1,3) GT).Being imported in HepG2 cell by this recombinant vector pLVX-Puro/GT, obtain containing α 1, the 3 related gene coded sequence of galactosyl transferase and the reconstitution cells of EGFP coded sequence, by named for this reconstitution cell HepG2 (GT+).
Wherein, the 1st-the 1630 nucleotide of SEQIDNo.4 is α 1, the nucleotide sequence of 3 galactosyl transferase related genes, and identical with the 1st of SEQIDNo.5 the-the 1630 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.3；The 581st-the 1630 nucleotide of SEQIDNo.4 is α 1,3 galactosyl transferase gene coded sequences, and identical with the 581st of SEQIDNo.5 the-the 1630 nucleotide, encoding amino acid sequence is the protein of SEQIDNo.3；The DNA molecular that 1st-the 548 nucleotide is entitled GTTI of SEQIDNo.4 is identical with the 1st of SEQIDNo.5 the-the 548 nucleotide；The 1646th-the 2365 nucleotide coding EGFP of SEQIDNo.4 is identical with the 3344th of SEQIDNo.5 the-the 4063 nucleotide.By in pLVX-Puro vector introduction HepG2 cell, obtain the reconstitution cell of pLVX-Puro carrier, by named for this reconstitution cell HepG2 (pLVX-Puro).
2, the qualification of reconstitution cell
Utilize the expression of Endoglin in HepG2 (GT-Eng+) in Westernblot method detecting step 1, one resists for Endoglin antibody (Abcam product, article No. is ab169545), two resist for horseradish peroxidase-labeled goat anti-rabbit igg, and DAB colour developing result is as shown in Figure 2.Result shows cancerous cell HepG2 (GT-Eng+) middle expression Endoglin albumen of recombinating.
The expression of alpha (1,3) GT in immuno-fluorescence assay HepG2 (GT-Eng+):
1) 1mg/mLISOLECTINGS-IB4:500 μ gISOLECTINGS-IB4 dissolves with 500 μ L diluents, and diluent (pH7.2) is containing 1.0mMCaCl2With the 0.01MPBS of 2mM Hydrazoic acid,sodium salt (sodiumazide), subpackage 5 μ L or 10 μ L every after dissolving ,-20 DEG C keep in Dark Place, it is to avoid multigelation.First dissolve with front, brief centrifugation, take supernatant 0.01MPBS and dilute 100 times, obtain the GS-IB4 of dilution, omnidistance lucifuge operation.
2) cultivating logarithmic (log) phase cell, be layered in the burnt special ware of copolymerization (culture dish at the bottom of 35mm glass, aperture 10mm, slide thickness 0.085-0.13mm), each ware adds 1 × 105Individual cell is layered in the aperture of centre.After cell attachment well-grown, from incubator, take out cell, abandon culture medium, PBS washes 2 times (carefully avoiding cell detachment), by the fixing cell of 4% paraformaldehyde (being paved with bottom copolymerization Jiao's ware), room temperature 20min, PBS washes 3 times, 3min/ time.
3) add 0.2%TritonX-100 (being paved with bottom copolymerization Jiao's ware) thoroughly to change 10min, 1 × PBS and wash 3 times, 3min/ time.
4) add 2% calf serum (being paved with bottom copolymerization Jiao's ware) to close, room temperature 30min.
5) abandon confining liquid PBS and wash 3 times, 3min/ time.Add step 1) the GS-IB4 (being paved with bottom copolymerization Jiao's ware) of dilution, 4 DEG C of wet boxes are hatched 3h, blank group adds PBS and replaces GS-IB4, this step lucifuge.PBS washes 3 times, 5min/ time, this step lucifuge.
6) flow cytomery average fluorescent strength is used.
According to the method described above, HepG2 (GT-Eng+) is replaced with respectively HepG2 (Eng+), HepG2 (GT+), HepG2 and HepG2 (pLVX-Puro), other steps are the most constant, respectively obtain the expression of results of alpha (1,3) GT in HepG2 (Eng+), HepG2 (GT+), HepG2 and HepG2 (pLVX-Puro).
Alpha (1,3) the GT catalysis expressed in HepG2 (GT-Eng+) generates α-Gal, α-Gal and AlexaThe agglutinin ISOLECTINGS-IB4 of 647 labellings combines, and be excited red fluorescence.Result shows (Fig. 3), and HepG2 (GT-Eng+) and HepG2 (GT+) all expresses alpha (1,3) GT.
3, the cultivation of reconstitution cell
Cultivate HepG2 and the HepG2 (GT-Eng+) of step 1, HepG2 (Eng+), HepG2 (GT+) and HepG2 (pLVX-Puro), the HepG2 respectively obtain the HepG2 being in logarithmic (log) phase, being in logarithmic (log) phase (GT-Eng+), be in the HepG2 (Eng+) of logarithmic (log) phase, be in the HepG2 (GT+) of logarithmic (log) phase and be in the HepG2 (pLVX-Puro) of logarithmic (log) phase.
Two, the preparation of dendritic cell
S1, the blood PBS taking people are diluted, blood after dilution is added in the centrifuge tube being placed with lymphocyte separation medium slowly (twice of volume that the volume of the blood after dilution is lymphocyte separation medium), make the blood after dilution slowly run down into lymphocyte separation medium skin lamination along tube wall, make the blood suspension after dilution on lymphocyte separation medium；
S2,2300rpm, centrifugal 30min, centrifuge raising speed and reduction of speed are 1 grade；
S3, liquid in pipe are layered, and are plasma layer the most successively, PBMC (PERIPHERAL BLOOD MONONUCLEAR CELL) layer, separate liquid, GCL, red blood cell layer.Handle with care, draw middle cloud tunica albuginea confluent monolayer cells with 1mL syringe, collect in new 50mL centrifuge tube, obtain PBMC liquid；
S4,37 DEG C of incomplete RIPM1640 (i.e. the RIPM1640 without the serum) washing of 4 times of PBMC liquid volumes of addition, 1500rpm, centrifugal 10min；Again with the washing of 37 DEG C of 4 times of PBMC liquid volumes infull RIPM1640 (i.e. the RIPM1640 without serum), 1100rpm, 10min；Again with the washing of 37 DEG C of 4 times of PBMC liquid volumes infull RIPM1640 (i.e. the RIPM1640 without serum), 1000rpm, centrifugal 10min；
S5, with the complete RIPM1640 RIPM1640 of serum (i.e. containing) re-suspended cell, be put in culture bottle.At 37 DEG C, 5%CO2Cultivate 2 hours the precursor (be designated as DC and cultivate the DC of the 0th day) of dendritic cell (DC) (attached cell be) in incubator, add 1000U/mLrhGM-CSF and 500U/mLrhIL-4, be placed in 37 DEG C, 5%CO2In incubator.Within in DC incubation every three days, change liquid by the complete RIPM1640 containing 1000U/mLrhGM-CSF, 500U/mLrhIL-4 half amount；
S6, cultivate the 5th day DC substantially in suspended state at DC, add the rhTNF-α of 25ng/mL, continue to be placed in 37%, 5%CO2Cultivate in incubator, cultivate the 7th day at DC and obtain ripe DC.The cellular morphology of this maturation DC is that volume becomes big, has root hair shape burr shape projection in colony growth, and form is irregular.
Measure above-mentioned mature dendritic cell (DC) through flow cytometer and express CD83, CD86, HL Α-DR and the level of HL Α-ABC.First by above-mentioned mature dendritic cell (DC) HLA-DR antibody (ebioscience product anti-with FITC-respectively, article No. is 11-9956-42), FITC-anti-HLA-ABC antibody (ebioscience product, article No. is 11-9983-41), FITC-anti-CD83 antibody (ebioscience product, article No. is 11-0839-42) and PE-anti-CD86 antibody (ebioscience product, article No. is 25-0869-42) react 45 minutes in PBS, reaction is washed three times with PBS after terminating, upper machine analysis.Result (Fig. 4) shows, ripe DC high expressed CD83, CD86, HLA-DR and HLA-ABC, cultivate compared with the DC (precursor of DC) of the 0th day with DC, the expression of CD83, CD86, HLA-DR has had significantly increases, and shows that the dendritic cell (DC) obtained in above-mentioned steps is ripe DC.
Three, the preparation of fused cell
The ripe DC of step 2 is washed one time with the RIPM1640 without serum by 3.1.Abandon supernatant, add 500 μ LDiluentC (NEB, B8003S) resuspended one-tenth individual cells suspensions.Separately add 1 μ LPKH26 stock solution (Sigma) mixing at 500 μ LDiluentC, add immediately after in cell, incubated at room 4min.Add 1mL calf serum and terminate 1min, with 6mL complete RIPM1640 culture medium wash three times standby, DC after being dyeed, above step needs lucifuge to carry out.
HepG2 (GT+) the tumor cell staining procedure being in logarithmic (log) phase of 3.2 steps 1: the tumor cell RIPM1640 without serum washes one time.Abandon supernatant, add the 1mL PBS resuspended one-tenth individual cells suspension containing 0.1% (mass percent concentration) BSA.Separately add 3 μ LFITC stock solutions (Sigma company) mixings at the 1mL PBS containing 0.1% (mass percent concentration) BSA, add immediately after in cell, hatch 8min, every 2min for 37 DEG C and shake once.Adding 1mL calf serum and terminate 1min, wash three times by 6mL complete RIPM1640 culture medium, the tumor cell after being dyeed, above step needs lucifuge to carry out.
Tumor cell after the dyeing of the DC after the dyeing of step 3.1 and step 3.2 is mixed by 3.3, and 1500rpm topples over supernatant after being centrifuged 10min, is not added with culture fluid and hatches 4min in 38 DEG C.Be slowly added at 38 DEG C the PEG2000250 μ L of preheating along centrifugal tube wall, during dropping will rotating centrifugal pipe slowly, make cell and PEG2000 be fully contacted.Then 38 DEG C, 4min is hatched.Slowly adding 40mLPBS along tube wall to terminate merging, 1200rpm, centrifugal 10min washs one time, adds the culture fluid complete DMEM containing 10% hyclone and is placed in 37 DEG C, 5%CO2Suspension cell is collected after cultivating 24h in incubator, Double fluorescence staining method as follows selects fused cell: detect the cell in above-mentioned cell suspension to be measured, the HepG2 (GT+) being in logarithmic (log) phase of step 1 only sends green fluorescence and does not send red fluorescence, the ripe DC of step 2 only sends red fluorescence and does not send green fluorescence, the fused cell of the two can send red fluorescence and send again green fluorescence, select and can send red fluorescence and send again the fused cell of green fluorescence, by named for this fused cell DC/HepG2 (GT+).Result shows, the percentage rate that fused cell DC/HepG2 (GT+) occurs is 65%.
According to the method described above, the HepG2 (GT+) being in logarithmic (log) phase being replaced with the HepG2 cell being in logarithmic (log) phase that step one obtains, other steps are constant, obtain the fused cell DC/HepG2 of HepG2 and DC.
According to the method described above, the HepG2 (GT+) being in logarithmic (log) phase is replaced with the HepG2 (pLVX-Puro) being in logarithmic (log) phase that step one obtains, other steps are constant, obtain the fused cell DC/HepG2 (pLVX-Puro) of HepG2 (pLVX-Puro) and DC.
According to the method described above, the HepG2 (GT+) being in logarithmic (log) phase is replaced with the HepG2 (GT-Eng+) being in logarithmic (log) phase that step one obtains, other steps are constant, obtain the fused cell DC/HepG2 (GT-Eng+) of HepG2 (GT-Eng+) and DC.
According to the method described above, the HepG2 (GT+) being in logarithmic (log) phase is replaced with the HepG2 (Eng+) being in logarithmic (log) phase that step one obtains, other steps are constant, obtain the fused cell DC/HepG2 (Eng+) of HepG2 (Eng+) and DC.
Embodiment 2, the generation of T lymphocyte of DC/HepG2 (GT-Eng+) external evoked secretion of gamma-IFN
Experiment in triplicate, repeats specifically comprising the following steps that of experiment every time
Obtain the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT+) induces by the following method, and with enzyme-linked immunospot assay (enzymelinkedimmunospotassay, ELISPOT) carry out detecting the IFN-γ of T lymphocytic emiocytosis, 1 × Washingbuffer used, biotin labeled antibody, enzyme mark Avidin is HumanIFN-gammaprecoatedELISPOTkit, and (HumanIFN-gammaprecoatedELISPOTkit is Bioisystech Co., Ltd's product for reaching section, article No. is DKW22-1000-048) in reagent, specifically comprise the following steps that
1) taking out the orifice plate in test kit, add the infull RIPM1640 culture medium of 200 μ L in every hole, room temperature stands 5-10 minute, is outwelled by liquid.
2) DC/HepG2 (GT+) 3 × 10 of the embodiment 1 suspended by infull RIPM1640 culture medium is all added to every hole4Individual and T lymphocyte 3 × 105Individual.Every hole 100 μ L amount of liquid, the often multiple hole of group 4.
3) hatch: build plate lid, put into incubator and cultivate 5 days, obtain the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT+) induces.
4) cleavage step 3) the T lymphocyte of secretion of gamma-IFN induced of DC/HepG2 (GT+): pouring aperture inner cell and culture medium.Adding the deionized water of 200 L4 DEG C of pre-coolings of μ in every hole, 15 minutes hypotonic lysis cells placed by 4 DEG C of refrigerators.
5) plate is washed: the liquid toppling in culture plate, the 200 μ L1 × Washingbuffer washing of the most every hole, wash altogether 5 times, stop the 60-80 second every time.For the last time, after pouring out the liquid in culture plate, culture plate is tipped upside down on no liquid in absorbent paper up to culture plate.
6) detection antibody incubation: by with sterilized water according to biotin labeled antibody: in every hole of biotin labeled antibody that the dilution proportion of sterilized water=1:1000 obtains addition culture plate, every hole 100 μ L.Hatch 1 hour for 37 DEG C.
7) wash plate: the liquid toppling in culture plate, add 1 × Washingbuffer, every hole 200 μ L, wash 5-6 time.Stop the 60-80 second every time.For the last time, in absorbent paper, button is dry.
8) enzyme connection Avidin is hatched: by with sterilized water according to enzyme mark Avidin: in every hole of enzyme mark Avidin that the dilution proportion of sterilized water=1:100 obtains addition culture plate, every hole 100 μ L.Hatch 1 hour for 37 DEG C.
9) wash plate: the liquid toppling in culture plate, add 1 × Washingbuffer, every hole 200 μ L, wash 5-6 time.Stop the 60-80 second every time.For the last time, in absorbent paper, button is dry.
10) colour developing: freshly prepared AEC nitrite ion working solution is added each experimental port, every hole 100 μ L.Room temperature lucifuge stands 15-50 minute.
11) color development stopping: topple over the liquid in culture plate, opens plate base, spends tap water and rinses culture plate positive and negative and base 5 times, color development stopping.
12) after plate dries, analysis of threshold is set with CTL instrument, and records speckle parameter, do statistical analysis.
nullAccording to the method described above，DC/HepG2 (GT+) is replaced with DC/HepG2 (GT-Eng+) respectively、DC/HepG2、DC/HepG2(Eng+)、DC/HepG2(pLVX-Puro)、DC、HepG2、HepG2(Eng+)、HepG2(GT+)、HepG2 (GT-Eng+) and HepG2 (pLVX-Puro)，Respectively obtain the T lymphocyte that DC/HepG2 (GT-Eng+) induces、The T lymphocyte of DC/HepG2 induction、The T lymphocyte that DC/HepG2 (Eng+) induces、The T lymphocyte that DC/HepG2 (pLVX-Puro) induces、The T lymphocyte of DC induction、The T lymphocyte of HepG2 induction、The T lymphocyte that HepG2 (Eng+) induces、The T lymphocyte that HepG2 (GT+) induces、T lymphocyte that HepG2 (GT-Eng+) induces and the T lymphocyte that HepG2 (pLVX-Puro) induces，And the ELISpot testing result of the amount of the IFN-γ of these T lymphocytic emiocytosis.
Result shows, the T lymphocyte secretion of gamma-IFN hardly that HepG2, HepG2 (Eng+), HepG2 (GT+), HepG2 (GT-Eng+) and HepG2 (pLVX-Puro) are external evoked；The speckle number of the IFN-γ of DC/HepG2 (GT-Eng+) inducer T lymphocyte secretion is above the speckle number of the IFN-γ of DC/HepG2 (GT+), DC/HepG2, DC/HepG2 (pLVX-Puro) and the secretion of DC inducer T lymphocyte.Showing, DC/HepG2 (GT-Eng+) can the generation of the efficient T lymphocyte of secretion inducing IFN-γ.
The treatment to tumor of the T lymphocyte of the secretion of gamma-IFN that embodiment 3, fused cell DC/HepG2 (GT-Eng+) induction produces
Experiment in triplicate, repeats specifically comprising the following steps that of experiment every time
nullBy the T lymphocyte of the secretion of gamma-IFN that the DC/HepG2 (GT+) of embodiment 2 induces、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces、The T lymphocyte of the secretion of gamma-IFN of DC/HepG2 induction、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces、The T lymphocyte of the secretion of gamma-IFN of DC induction、The T lymphocyte of HepG2 induction、The T lymphocyte that HepG2 (Eng+) induces、T lymphocyte that HepG2 (GT+) induces and the T lymphocyte that HepG2 (pLVX-Puro) induces are suspended in PBS respectively，Respectively obtain cell content and be 105nullThe T Lymphocyte suspension of the secretion of gamma-IFN that the DC/HepG2 (GT+) of individual/μ L induces、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces、The T Lymphocyte suspension of the secretion of gamma-IFN of DC/HepG2 induction、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces、The T Lymphocyte suspension of the secretion of gamma-IFN of DC induction、The T Lymphocyte suspension of HepG2 induction、The T Lymphocyte suspension that HepG2 (Eng+) induces、T Lymphocyte suspension that HepG2 (GT+) induces and the T Lymphocyte suspension that HepG2 (pLVX-Puro) induces.
Taking 20 4-5 week old inbred line Female nude mice (BALB/cNudeMice), every in oxter, right side subcutaneous vaccination 5 × 106Individual HepG2 hepatoma carcinoma cell.Measure tumor major diameter and minor axis twice a week, according to formula TV=1/2 × a × b2Calculate gross tumor volume.Treat that tumor average volume length is to about 100mm3Time, (in the T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (GT+) induces, the content of T lymphocyte is 10 to inject the T Lymphocyte suspension of the secretion of gamma-IFN that 100 μ LDC/HepG2 (GT+) induce in the vein to every lotus tumor BalB/c nude mice7Individual/100 μ L) treat, it is designated as treating the 0th day by injection for the first time, the T Lymphocyte suspension of the secretion of gamma-IFN that the above-mentioned DC/HepG2 of 100 μ L (GT+) induces within 36th day, is all injected respectively in treatment the 6th day, treatment the 12nd day, treatment the 18th day, treatment the 24th, treatment the 30th day and treatment, the volume of lotus tumor BalB/c nude mouse tumor is measured respectively before the injection on the per injection same day, within 1st day, start to record the time-to-live of every mice from treatment, the survival rate of statistics mice.
nullAccording to the method described above，The T Lymphocyte suspension of the secretion of gamma-IFN induced by DC/HepG2 (GT+) respectively replaces with PBS、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces、The T Lymphocyte suspension of the secretion of gamma-IFN of DC/HepG2 induction、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces、The T Lymphocyte suspension of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces、The T Lymphocyte suspension of the secretion of gamma-IFN of DC induction、The T Lymphocyte suspension of HepG2 induction、The T Lymphocyte suspension that HepG2 (Eng+) induces、T Lymphocyte suspension that HepG2 (GT+) induces and the T Lymphocyte suspension that HepG2 (pLVX-Puro) induces，Other steps are the most constant，Respectively obtain PBS、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces、The T lymphocyte of the secretion of gamma-IFN of DC/HepG2 induction、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces、The T lymphocyte of the secretion of gamma-IFN that DC induction produces、The T lymphocyte of HepG2 induction、The T lymphocyte that HepG2 (Eng+) induces、T lymphocyte that HepG2 (GT+) induces and the T lymphocyte that HepG2 (pLVX-Puro) induces therapeutic effect (Fig. 5 to tumor、Tables 1 and 2).
Table 1, different cell induction T lymphocyte treatment tumor after the volume (mm of tumor3)
nullWhen processing the 36th day，The volume of the tumor after the T lymphocyte treatment of the secretion of gamma-IFN induced through DC/HepG2 (GT-Eng+) is respectively PBS、The T lymphocyte of the secretion of gamma-IFN of DC/HepG2 induction、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (Eng+) induces、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT+) induces、The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (pLVX-Puro) induces、The T lymphocyte of the secretion of gamma-IFN of DC induction、The T lymphocyte of HepG2 induction、The T lymphocyte that HepG2 (Eng+) induces、T lymphocyte that HepG2 (GT+) induces and 0.079 times of the T lymphocyte that HepG2 (pLVX-Puro) induces、0.111 times、0.276 times、0.274 times、0.131 times、0.107 times、0.070 times、0.068 times、0.072 times、0.074 times.
Result shows, the T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces can significantly delay tumor growth, the T lymphocyte that effect is induced significantly better than DC/HepG2 (GT+), DC/HepG2 (Eng+), DC/HepG2 (pLVX-Puro), DC/HepG2, DC, HepG2, HepG2 (Eng+), HepG2 (GT+) and HepG2 (pLVX-Puro) treatment to tumor.
Table 2, lotus people's Liver Cancer Bearing Nude Mice when different survival rates away from the 1st time treatment time (my god)
| Survival rate | 80% | 60% | 40% | 20% | 0 |
| PBS | 98 | 114 | 126 | 143 | 152 |
| DC/HepG2 | 132 | 163 | 167 | 178 | 187 |
| DC/HepG2(GT-Eng+) | 217 | 225 | 240 | —— | —— |
| DC/HepG2(Eng+) | 165 | 174 | 189 | 217 | 226 |
| DC/HepG2(GT+) | 187 | 189 | 198 | 206 | 235 |
| DC/HepG2(pLVX-Puro) | 152 | 153 | 163 | 176 | 182 |
| DC | 145 | 158 | 160 | 168 | 171 |
| HepG2 | 84 | 95 | 96 | 101 | 110 |
| HepG2(Eng+) | 96 | 97 | 102 | 104 | 112 |
| HepG2(GT+) | 89 | 93 | 97 | 110 | 111 |
| HepG2(pLVX-Puro) | 91 | 94 | 95 | 109 | 113 |
Note: " " represents these data of undetermined.
nullResult shows，The T lymphocyte of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) induces can be obviously prolonged lotus people's Liver Cancer Bearing Nude Mice life span: with PBS、DC/HepG2(GT+)、DC/HepG2(Eng+)、DC/HepG2(pLVX-Puro)、DC/HepG2、DC、HepG2、HepG2(Eng+)、HepG2(GT+)、The T lymphocyte that HepG2 (GT-Eng+) and HepG2 (pLVX-Puro) induces is compared，Survival rate at lotus people's Liver Cancer Bearing Nude Mice is respectively 80%、When 60% and 40%，The lotus people's Liver Cancer Bearing Nude Mice of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) the induces time away from the 1st treatment all extends；When the survival rate of lotus people's Liver Cancer Bearing Nude Mice is 40%, the lotus people's Liver Cancer Bearing Nude Mice of the T lymphocyte treatment of the secretion of gamma-IFN that DC/HepG2 (GT-Eng+) the induces time away from the 1st treatment is respectively 1.21 times of T lymphocyte, 1.27 times, 1.47 times, 1.44 times, 1.5 times, 2.5 times, 2.35 times, 2.47 times, 2.53 times that DC/HepG2 (GT+), DC/HepG2 (Eng+), DC/HepG2 (pLVX-Puro), DC/HepG2, DC, HepG2, HepG2 (Eng+), HepG2 (GT+) and HepG2 (pLVX-Puro) induce.During treatment and observing, mice is all without piloerection, appetite decline, the phenomenons such as behavioral activity is abnormal.
The T lymphocyte of the secretion of gamma-IFN that the DC/HepG2 (GT-Eng+) of the application induces can substantially suppress tumor growth, extends lotus people's Liver Cancer Bearing Nude Mice life span.
Claims (10)
1. the method preparing fused cell, for S1 or S2:
S1, described fused cell are fused cell 1；The described method preparing fused cell 1 includes carrying out tumor cell 1 and dendritic cell merging the step obtaining fused cell 1；
Described tumor cell 1 is M or N:
M, described tumor cell 1 are for expressing Endoglin and α 1, the reconstitution cell of 3 galactosyl transferases；
N, described tumor cell 1 are containing gene and α 1, the reconstitution cell of 3 galactosyl transferase related genes encoding described Endoglin；
Described Endoglin is following A 1) or protein A2):
A1) aminoacid sequence is the protein of SEQIDNo.1；
A2) in the aminoacid sequence shown in SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) derivative protein；
Described α 1,3 galactosyl transferase is following B1) or protein B2):
B1) aminoacid sequence is the protein of SEQIDNo.3；
B2) in the aminoacid sequence shown in SEQIDNo.3 through replacement and/or disappearance and/or add that one or several amino acid residue obtains there is α 1,3 galactosyl transferase functions by B1) derivative protein；
Described α 1,3 galactosyl transferase related gene is formed by connecting by α 1, the DNA molecular of 3 galactosyl transferase genes and entitled GTTI；
α 1,3 galactosyl transferase described in described α 1,3 galactosyl transferase gene code；
The nucleotides sequence of described GTTI is classified as the 1st-the 548 nucleotide of SEQ ID No .5；
S2, described fused cell are fused cell 2；The described method preparing fused cell 2 includes carrying out tumor cell 2 and dendritic cell merging the step obtaining fused cell 2；
Described tumor cell 2 is that 3 galactosyl transferase genes also express described Endoglin and the reconstitution cell of described α 1,3 galactosyl transferase containing gene and the described α 1 encoding described Endoglin.
Method the most according to claim 1, it is characterised in that: described tumor cell 1 is that encoding gene and described α 1, the 3 galactosyl transferase related gene of described Endoglin are imported the reconstitution cell obtained in receptor tumor cells；
Described tumor cell 2 is the reconstitution cell that will obtain in receptor tumor cells described in the encoding gene of described Endoglin and described α 1,3 galactosyl transferase channel genes.
Method the most according to claim 1 and 2, it is characterised in that: the encoding gene of described Endoglin is following A11) A21) or A31) shown in nucleic acid molecules:
A11) the 1640th-the 3322 DNA molecular shown in nucleotide of SEQ ID No .5；
A21) nucleotide sequence and A11) limited has 75% or more than 75% homogeneity, and encodes cDNA molecule or the genomic DNA molecule of described Endoglin；
A31) under strict conditions with A11) nucleotide sequence hybridization that limits, and encode cDNA molecule or the genomic DNA molecule of described Endoglin；
Described α 1,3 galactosyl transferase gene is following B11) or B21) or B31) shown in nucleic acid molecules:
B11) the 581st-the 1630 DNA molecular shown in nucleotide of SEQ ID No .5；
B21) nucleotide sequence and B11) limited has 75% or more than 75% homogeneity, and encodes cDNA molecule or the genomic DNA molecule of described α 1,3 galactosyl transferase；
B31) under strict conditions with B11) nucleotide sequence hybridization that limits, and encode cDNA molecule or the genomic DNA molecule of described α 1,3 galactosyl transferase.
4. according to the method described in claim 1 or 2 or 3, it is characterised in that: the nucleotides sequence of described α 1,3 galactosyl transferase related gene is classified as the 1st-the 1630 nucleotide of SEQ ID No .5；
Described tumor cell 1 is that the DNA molecular shown in the 1st of SEQIDNo.5 the-the 3322 nucleotide is imported the reconstitution cell obtained in described receptor tumor cells；
Described tumor cell 2 for importing the reconstitution cell obtained in described receptor tumor cells by the DNA molecular shown in the 581st of SEQIDNo.5 the-the 3322 nucleotide.
5. according to described method arbitrary in claim 1-4, it is characterised in that: described receptor tumor cells is hepatoma carcinoma cell.
6. the fused cell that in claim 1-5, arbitrary described method obtains.
7. treatment and/or prophylaxis of tumours medicine, it is characterised in that: the active component of described treatment and/or prophylaxis of tumours medicine is following H1 and/or H2:
Fused cell described in H1, claim 6；
The T lymphocyte of the secretion of gamma-IFN that fused cell inducer T lymphocyte described in H2, claim 6 obtains.
Any one product in the most following X1-X9:
X1, fusion protein, for following X1a) or protein X1b):
X1a) by the protein of the 549th of SEQIDNo.5 the-the 3281 nucleotide；
X1b) at X1a) protein aminoacid sequence in through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by X1a) derivative protein；
The encoding gene of fusion protein described in X2, X1, for the 1st-the 3322 nucleotide of SEQIDNo.5；
The encoding gene of fusion protein described in X3, X1, for the 581st-the 3322 nucleotide of SEQIDNo.5；
X4, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by described Endoglin composition arbitrary in described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4 and claim 1-3；
X5, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by the encoding gene composition of arbitrary described Endoglin in described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4 and claim 1-3；
X6, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by described Endoglin composition arbitrary in described α 1,3 galactosyl transferase gene arbitrary in claim 1-3 and claim 1-3；
X7, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by the encoding gene composition of arbitrary described Endoglin in described α 1,3 galactosyl transferase gene arbitrary in claim 1-3 and claim 1-3；
X8, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by described Endoglin composition arbitrary in described α 1,3 galactosyl transferase arbitrary in claim 1-3 and claim 1-3；
X9, for preparing treatment and/or the product of prophylaxis of tumours medicine, its active component is by the encoding gene composition of arbitrary described Endoglin in described α 1,3 galactosyl transferase arbitrary in claim 1-3 and claim 1-3.
The application of the most following N1 or N2 or N3 or N4 or N5:
The application in preparation treatment and/or prophylaxis of tumours medicine of the fused cell described in N1, claim 6 or the application in the T lymphocyte of preparation secretion of gamma-IFN；
The application in preparation treatment and/or prophylaxis of tumours medicine of the product described in N2, claim 8；
The application treated and/or in prophylaxis of tumours medicine prepared by N3 described Endoglin relevant biomaterial arbitrary to claim 1-3 and the biomaterial that arbitrary described α 1,3 galactosyl transferase related gene is relevant with claim 1-4；
Described described Endoglin relevant biomaterial arbitrary to claim 1-3 is following E1) to E20) in any one:
E1) nucleic acid molecules of arbitrary described Endoglin in coding claim 1-3；
E2) containing E1) expression cassette of described nucleic acid molecules；
E3) containing E1) recombinant vector of described nucleic acid molecules；
E4) containing E2) recombinant vector of described expression cassette；
E5) containing E1) recombinant microorganism of described nucleic acid molecules；
E6) containing E2) recombinant microorganism of described expression cassette；
E7) containing E3) recombinant microorganism of described recombinant vector；
E8) containing E4) recombinant microorganism of described recombinant vector；
E9) containing E1) the transgenetic animal cell system of described nucleic acid molecules；
E10) containing E2) the transgenetic animal cell system of described expression cassette；
E11) containing E3) the transgenetic animal cell system of described recombinant vector；
E12) containing E4) the transgenetic animal cell system of described recombinant vector；
E13) containing E1) the transgenic animal tissue of described nucleic acid molecules；
E14) containing E2) the transgenic animal tissue of described expression cassette；
E15) containing E3) the transgenic animal tissue of described recombinant vector；
E16) containing E4) the transgenic animal tissue of described recombinant vector；
E17) containing E1) transgenic animal organ of described nucleic acid molecules；
E18) containing E2) transgenic animal organ of described expression cassette；
E19) containing E3) transgenic animal organ of described recombinant vector；
E20) containing E4) transgenic animal organ of described recombinant vector；
Described with claim 1-4 in the relevant biomaterial of arbitrary described α 1,3 galactosyl transferase related gene be following F1) to F19) and in any one:
F1) expression cassette containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F2) recombinant vector containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F3) containing F1) recombinant vector of described expression cassette；
F4) recombinant microorganism containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F5) containing F1) recombinant microorganism of described expression cassette；
F6) containing F2) recombinant microorganism of described recombinant vector；
F7) containing F3) recombinant microorganism of described recombinant vector；
F8) the transgenetic animal cell system containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F9) containing F1) the transgenetic animal cell system of described expression cassette；
F10) containing F2) the transgenetic animal cell system of described recombinant vector；
F11) containing F3) the transgenetic animal cell system of described recombinant vector；
F12) the transgenic animal tissue containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F13) containing F1) the transgenic animal tissue of described expression cassette；
F14) containing F2) the transgenic animal tissue of described recombinant vector；
F15) containing F3) the transgenic animal tissue of described recombinant vector；
F16) transgenic animal organ containing described α 1,3 galactosyl transferase related gene arbitrary in claim 1-4；
F17) containing F1) transgenic animal organ of described expression cassette；
F18) containing F2) transgenic animal organ of described recombinant vector；
F19) containing F3) transgenic animal organ of described recombinant vector；
N4 described Endoglin relevant biomaterial arbitrary to claim 1-3 and the biomaterial of arbitrary described α 1, the 3 galactosyl transferase gene-correlation application in preparation treatment and/or prophylaxis of tumours medicine with claim 1-3；
Described with claim 1-3 in the biomaterial of arbitrary described α 1,3 galactosyl transferase gene-correlation be following G1) to G19) and in any one:
G1) expression cassette containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G2) recombinant vector containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G3) containing G1) recombinant vector of described expression cassette；
G4) recombinant microorganism containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G5) containing G1) recombinant microorganism of described expression cassette；
G6) containing G2) recombinant microorganism of described recombinant vector；
G7) containing G3) recombinant microorganism of described recombinant vector；
G8) the transgenetic animal cell system containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G9) containing G1) the transgenetic animal cell system of described expression cassette；
G10) containing G2) the transgenetic animal cell system of described recombinant vector；
G11) containing G3) the transgenetic animal cell system of described recombinant vector；
G12) the transgenic animal tissue containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G13) containing G1) the transgenic animal tissue of described expression cassette；
G14) containing G2) the transgenic animal tissue of described recombinant vector；
G15) containing G3) the transgenic animal tissue of described recombinant vector；
G16) transgenic animal organ containing described α 1,3 galactosyl transferase gene arbitrary in claim 1-3；
G17) containing G1) transgenic animal organ of described expression cassette；
G18) containing G2) transgenic animal organ of described recombinant vector；
G19) containing G3) transgenic animal organ of described recombinant vector；
The application in preparation treatment and/or prophylaxis of tumours medicine of the T lymphocyte of the secretion of gamma-IFN that fused cell inducer T lymphocyte described in N5, claim 6 obtains.
The most following P1)-P7) in any one treatment and/or prophylaxis of tumours medicine:
P1) treatment and/or prophylaxis of tumours medicine prepared by the fused cell described in claim 6, are utilized；
Treatment prepared by the biomaterial P2), utilizing α 1, the 3 galactosyl transferase related gene described in biomaterial relevant for the Endoglin described in claim 9 and claim 9 relevant and/or prophylaxis of tumours medicine；
P3) treatment and/or prophylaxis of tumours medicine prepared by the biomaterial of α 1, the 3 galactosyl transferase gene-correlation described in biomaterial relevant for the Endoglin described in claim 9 and claim 9, are utilized；
P4) treatment and/or prophylaxis of tumours medicine prepared by the product described in claim 8, are utilized；
P5) treatment and/or the prophylaxis of tumours medicine of arbitrary described tumor cell 1 preparation in claim 1-5, are utilized；
P6) treatment and/or the prophylaxis of tumours medicine of arbitrary described tumor cell 2 preparation in claim 1-5, are utilized；
P7) treatment and/or prophylaxis of tumours medicine prepared by the T lymphocyte of the secretion of gamma-IFN that fused cell inducer T lymphocyte described in claim 6 obtains, are utilized.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510001075.6A CN105821029A (en) | 2015-01-04 | 2015-01-04 | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510001075.6A CN105821029A (en) | 2015-01-04 | 2015-01-04 | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105821029A true CN105821029A (en) | 2016-08-03 |
Family
ID=56986801
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510001075.6A Pending CN105821029A (en) | 2015-01-04 | 2015-01-04 | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105821029A (en) |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328569A (en) * | 1998-11-24 | 2001-12-26 | 纳瓦拉公司科学与技术研究所 | TGF (b) 1 inhibitor peptides |
| CN1427889A (en) * | 2000-01-11 | 2003-07-02 | 格林维尔医院系统公司 | Hybrid cells obtainable from antigen presenting cells |
| CN1485423A (en) * | 2002-09-27 | 2004-03-31 | 上海博德基因开发有限公司 | Polypeptide-human galactosyltransferase 42 and polynucleotide encoding the polypeptide |
| CN1704477A (en) * | 2004-05-31 | 2005-12-07 | 杨捷 | Alpha (1,3) galactose transferase recombinant cattier and its production and use |
| WO2007002264A2 (en) * | 2005-06-22 | 2007-01-04 | The Johns Hopkins University | Biomarker for ovarian cancer: ctap3-related proteins |
| CN1891829A (en) * | 2005-07-05 | 2007-01-10 | 严泉剑 | Alpha (1,3) galactosyl transferase recombinant vector, virosome and its preparation and use |
| CN101402686A (en) * | 2008-10-20 | 2009-04-08 | 中国人民解放军第三军医大学 | Antihuman Endoglin single-chain antibody, preparation and uses thereof |
| WO2009074341A1 (en) * | 2007-12-12 | 2009-06-18 | Trimed Biotech Gmbh | Method for producing dendritic cells |
| WO2010032059A2 (en) * | 2008-09-19 | 2010-03-25 | Medimmune Llc | Targeted binding agents directed to cd105 and uses thereof |
| CN101932339A (en) * | 2007-11-30 | 2010-12-29 | 贝勒医学院 | Dendritic cell vaccine composition and its application |
| WO2011041441A1 (en) * | 2009-09-30 | 2011-04-07 | Tracon Pharmaceuticals, Inc. | Endoglin antibodies |
| CN102258789A (en) * | 2010-12-29 | 2011-11-30 | 海南医学院 | Miniaturized Endoglin antibody and adriamycin conjugate and preparation method thereof |
| CN102625714A (en) * | 2008-07-16 | 2012-08-01 | 贝勒研究院 | HIV vaccine based on targeting maximized GAG and NEF to dendritic cells |
| CN102839157A (en) * | 2012-07-12 | 2012-12-26 | 扬州大学 | MDCK cell system capable of stably coexpressing Hst3GIIV and beta1-4GalT1 and establishing method and application thereof |
| WO2013092001A1 (en) * | 2011-12-19 | 2013-06-27 | Synimmune Gmbh | Bispecific antibody molecule |
-
2015
- 2015-01-04 CN CN201510001075.6A patent/CN105821029A/en active Pending
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328569A (en) * | 1998-11-24 | 2001-12-26 | 纳瓦拉公司科学与技术研究所 | TGF (b) 1 inhibitor peptides |
| CN1427889A (en) * | 2000-01-11 | 2003-07-02 | 格林维尔医院系统公司 | Hybrid cells obtainable from antigen presenting cells |
| CN1485423A (en) * | 2002-09-27 | 2004-03-31 | 上海博德基因开发有限公司 | Polypeptide-human galactosyltransferase 42 and polynucleotide encoding the polypeptide |
| CN1704477A (en) * | 2004-05-31 | 2005-12-07 | 杨捷 | Alpha (1,3) galactose transferase recombinant cattier and its production and use |
| WO2007002264A2 (en) * | 2005-06-22 | 2007-01-04 | The Johns Hopkins University | Biomarker for ovarian cancer: ctap3-related proteins |
| CN1891829A (en) * | 2005-07-05 | 2007-01-10 | 严泉剑 | Alpha (1,3) galactosyl transferase recombinant vector, virosome and its preparation and use |
| CN101932339A (en) * | 2007-11-30 | 2010-12-29 | 贝勒医学院 | Dendritic cell vaccine composition and its application |
| WO2009074341A1 (en) * | 2007-12-12 | 2009-06-18 | Trimed Biotech Gmbh | Method for producing dendritic cells |
| CN102625714A (en) * | 2008-07-16 | 2012-08-01 | 贝勒研究院 | HIV vaccine based on targeting maximized GAG and NEF to dendritic cells |
| WO2010032059A2 (en) * | 2008-09-19 | 2010-03-25 | Medimmune Llc | Targeted binding agents directed to cd105 and uses thereof |
| CN101402686A (en) * | 2008-10-20 | 2009-04-08 | 中国人民解放军第三军医大学 | Antihuman Endoglin single-chain antibody, preparation and uses thereof |
| WO2011041441A1 (en) * | 2009-09-30 | 2011-04-07 | Tracon Pharmaceuticals, Inc. | Endoglin antibodies |
| CN102258789A (en) * | 2010-12-29 | 2011-11-30 | 海南医学院 | Miniaturized Endoglin antibody and adriamycin conjugate and preparation method thereof |
| WO2013092001A1 (en) * | 2011-12-19 | 2013-06-27 | Synimmune Gmbh | Bispecific antibody molecule |
| CN102839157A (en) * | 2012-07-12 | 2012-12-26 | 扬州大学 | MDCK cell system capable of stably coexpressing Hst3GIIV and beta1-4GalT1 and establishing method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 孟繁平等主编: "《临床免疫基础》", 31 August 2004, 郑州大学出版社 * |
| 张利旺: "全细胞性肿瘤抗原以不同方式负载树突状细胞诱导肝癌特异性T细胞反应的实验研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 张天一等: "Endoglin与肿瘤血管生成、浸润及转移之间的关系", 《现代肿瘤医学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Diebold | Determination of T‐cell fate by dendritic cells | |
| Okada et al. | Augmentation of the migratory ability of DC-based vaccine into regional lymph nodes by efficient CCR7 gene transduction | |
| CA3011933A1 (en) | Oncolytic viral vectors and uses thereof | |
| JP6841812B2 (en) | Agents and compositions for eliciting an immune response | |
| Ramachandran et al. | Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration | |
| CN113151285B (en) | Human 4IgB7-H3 mutant coding gene and its application in regulating immunity | |
| Furugaki et al. | Intraperitoneal administration of a tumor-associated antigen SART3, CD40L, and GM-CSF gene-loaded polyplex micelle elicits a vaccine effect in mouse tumor models | |
| del Rio et al. | CX3CR1+ c-kit+ bone marrow cells give rise to CD103+ and CD103− dendritic cells with distinct functional properties | |
| CN109069604A (en) | Neoantigen composition and its application method in immune oncotherapy | |
| Lin et al. | F4/80: the macrophage-specific adhesion-GPCR and its role in immunoregulation | |
| CN109415706A (en) | The oncolytic rhabdovirus of pseudotyping and its application in combined therapy | |
| González et al. | Melanoma cell lysate induces CCR 7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells | |
| Christensen et al. | A critical role of the PTEN/PDGF signaling network for the regulation of radiosensitivity in adenocarcinoma of the prostate | |
| Walzer et al. | Poxvirus semaphorin A39R inhibits phagocytosis by dendritic cells and neutrophils | |
| Li et al. | The combination of gemcitabine and ginsenoside Rh2 enhances the immune function of dendritic cells against pancreatic cancer via the CARD9-BCL10-MALT1/NF-κB pathway | |
| Jiang et al. | Immunotherapy with dendritic cells modified with tumor-associated antigen gene demonstrates enhanced antitumor effect against lung cancer | |
| CN103933558B (en) | A kind of preparation and application of therapeutic tumor vaccine of novel, wide spectrum | |
| Gao et al. | Immune cell recruitment and cell-based system for cancer therapy | |
| Komaru et al. | Sustained and NK/CD4+ T cell-dependent efficient prevention of lung metastasis induced by dendritic cells harboring recombinant Sendai virus | |
| Shi et al. | In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for malignant melanoma | |
| CN105821029A (en) | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof | |
| JP5049011B2 (en) | Increased T cell tumor invasion by mutant LIGHT | |
| CN109890834A (en) | Protein expression enhancer sequence and application thereof | |
| CN105821030A (en) | Cancer cell/dendritic cell fusion tumor vaccine for expression of alpha1,3 galactosyl transferase and preparation method thereof | |
| Li et al. | Dual‐Targeted Engineered Bacterial Outer Membrane Vesicles for Hepatocellular Carcinoma Immunotherapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination |