CN102552891A - Preparation method of heat shock apoptosis glioma-loaded dendritic cell tumor vaccine - Google Patents
Preparation method of heat shock apoptosis glioma-loaded dendritic cell tumor vaccine Download PDFInfo
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Abstract
The invention provides a preparation method of a heat shock apoptosis glioma-loaded dendritic cell tumor vaccine. The method comprises the following steps of: setting a tumor cell in a heat shock state; inducing a cell high-expression heat shock protein (HSP); inducing cell apoptosis by using a chemical medicament to obtain an apoptosis cell antigen which is rich in HSPs; and culturing the prepared antigen with an immature dendritic cell (DC) in a hybrid way to obtain a mature DC vaccine. A heat shock glioma cell-loaded DC vaccine has high apoptosis rate, and has the phenotypic traits of a mature DC as proved by flow cytometry detection, the average value of universally-recognized marks HLA-DR<+>CD11C<+> of the DC is 90.90 percent, the average value of mature marks CD11c<+>CD86<+> is 73.03 percent, the average value of CD11c<+>CD83<+> is 86.04 percent, and CD83 and CD86 are expressed efficiently. The DC vaccine prepared by loading with a heat shock method has high maturity, and can be used for inducing immune response.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of method for preparing of heat shock apoptosis cerebral glioma loaded dendritic cell tumor vaccine.
Background technology
The brain glioblastoma is a most common tumor in central nervous system's primary malignant tumor.Because of fast growth and along the nerve fiber infiltrative growth, cause operation to be difficult to excise fully; Simultaneously chemotherapy and radiation is merely medium sensitivity.Therefore, the sequential therapy scheme of existing internationally recognized routine operation, radiotherapy, chemotherapy is difficult to effect a radical cure the brain glioblastoma at present.Immunization therapy is through adjustment, human body immunity improving systemic-function, removes residual tumor cell, reaches the purpose of real healing.At present, be that basic cell vaccine treatment is a kind of active immunity treatment with DC, just becoming the focus of research.DC is an antigen presenting cell (antigen presenting cell APC) the most powerful in the body; Its angtigen presentation ability can be hundreds of times of other presenting cells; The T cell of ability effective stimulus tranquillization brings out primary immune response; It plays an important role in the identifying of T cell to tumor antigen, and DC can get into the targeting district through blood brain barrier, thus performance specificity antineoplastic effect.The In vitro culture of DC is being at present a Biotherapeutics technology commonly used, and the common method of animal and clinical experimental study can be divided into four types both at home and abroad: 1) specific tumour antigenic peptides load DC.The MHC-I class antigenic peptides that tumor antigen peptide can pass through synthetic, weak acid eluting tumor surface obtains; 2) tumor-cell antigen-loaded DC.Utilize methods such as ultrasonic disruption, multigelation or lonizing radiation irradiation tumor cell to obtain tumor cell property antigen, then the vaccine of sensitization DC preparation; 3) tumor cell-DC fusant vaccine.Hybrid cell after DC and tumor cell merge can obtain the phenotypic characteristic of this cell of parents; 4) tumor cell RNA or cDNA load DC.How selecting a kind of efficient, safe DC vaccine production method is that initiatively cellular immunization is treated key of success, significant for clinical generalization value.This method belongs to second type; Earlier tumor cell is in the heat shock state; Inducing cell high expressed heat shock protein (heat shock proteins HSP); Reuse chemicals cell death inducing and then be prepared into the apoptotic cell antigen that is rich in heat shock protein, antigen for preparing and immature DC mixing with cells are cultivated the sophisticated DC vaccine of preparation.
Summary of the invention
For realizing the object of the invention; The method for preparing of heat shock apoptosis cerebral glioma loaded dendritic cell tumor vaccine of the present invention adopts and earlier tumor cell is in the heat shock state; Inducing cell high expressed heat shock protein (HSP); Reuse chemicals cell death inducing and then be prepared into the apoptotic cell antigen that is rich in heat shock protein, antigen for preparing and immature DC mixing with cells are cultivated and are obtained sophisticated DC vaccine.The DC vaccine of heat shock brain glioblastoma cell load has apoptosis rate preferably, and through Flow cytometry, the DC vaccine has the phenotypic characteristic of ripe DC, the generally acknowledged sign HLA-DR of DC
+CD11c
+Meansigma methods be 90.90%, the maturation sign CD11c of DC
+CD86
+Meansigma methods be 73.03%, CD11c
+CD83
+Meansigma methods be 86.04%, high expressed CD83, CD86.Show that the DC vaccine Maturity that adopts the load of heat shock method to prepare is high, can be used for induce immune response.
Concrete, described method comprises the steps:
S1. the preparation of heat shock apoptosis brain glioblastoma cell and detection:
Human glioma cell line U251 draws the ATCC from the U.S., by the conventional method cultivation of going down to posterity.With RPMI 1640 culture medium adjustment cell concentration to 10
6/ ml places 40 ℃ with cell, 5%CO
23h in the incubator takes out Tissue Culture Flask, and adding betulinic acid to final concentration is 10 μ g/ml, Tissue Culture Flask is moved to 37 ℃, 5%CO
2Incubator continues to cultivate cell death inducing.The results apoptotic cell adds aseptic PBS washing 4 times, obtains apoptotic tumor cell antigen.
Tumor cell carries out streaming detection computations apoptosis rate result through 40 ℃ after marking cell with FITC-Annexin-V with PI pair after handling 3h.
Soft agarose cloning technique detects the external one-tenth tumor of cellular antigens ability, and the result shows negative for tumor cells clonal growth in the apoptotic cell; Endotoxin detects and is undertaken by the method for 2005 editions the 3rd regulations of the Pharmacopoeia of the People's Republic of China; Get preparation antigen and carry out the endotoxin detection.
The preparation of S2.DC tumor vaccine and detection
Gather 3 volunteer peripheral bloods, the peripheral blood that adopts artificial anticoagulant heparin to gather places to be transported to laboratory on ice; 2,000r/min, 10min; 20 ℃ obtain hemocytees, dilute 2 times after, Ficoll partition method washed corpuscles obtains once fresh PMNC.Be laid on behind the PBMC with the resuspended prepared fresh of culture medium in the six porocyte culture plates, be positioned over 37 ℃, 5%CO
2Behind the 90min, flush away is attached cell not in the incubator, in culture plate, adds and contains 100ng/mL GM-CSF; RPMI 1640 complete mediums of 50ng/mL IL-4; 3d changes liquid, and 5d gathers in the crops immature DC (imDC), adds apoptotic tumor cell antigen (the antigenic ratio of DC and target cell is 3: 1); To 37 ℃, 5%CO
2Incubator is cultivated 48h jointly, results DC cell, and tongue expects that cell counting is carried out in blue dyeing and phenotype detects.
Description of drawings
Through the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become obvious.Wherein:
Shown in Figure 1 is the steps flow chart sketch map of the method for preparing of heat shock apoptosis cerebral glioma loaded dendritic cell tumor vaccine of the present invention.
The specific embodiment
The steps flow chart sketch map of the method for preparing of heat shock apoptosis cerebral glioma loaded dendritic cell tumor vaccine of the present invention as shown in Figure 1, the concrete steps of said embodiment are following:
S1. the preparation of heat shock apoptosis brain glioblastoma cell and detection:
Human glioma cell line U251 draws the ATCC from the U.S., by the conventional method cultivation of going down to posterity.With RPMI1640 culture medium adjustment cell concentration to 10
6/ ml places 40 ℃ with cell, 5%CO
23h in the incubator takes out Tissue Culture Flask, and adding betulinic acid to final concentration is 10 μ g/ml, Tissue Culture Flask is moved to 37 ℃, 5%CO
2Incubator continues to cultivate cell death inducing.The results apoptotic cell adds aseptic PBS washing 4 times, obtains apoptotic tumor cell antigen.
Tumor cell carries out streaming detection computations apoptosis rate result demonstration after marking cell with FITC-Annexin-V and PI are two after handling 3h through 40 ℃, and the apoptosis rate meansigma methods is 66.23%.
Soft agarose cloning technique detects the external one-tenth tumor of cellular antigens ability, and the result shows negative for tumor cells clonal growth in the apoptotic cell.Endotoxin detects and is undertaken by the method for 2005 editions the 3rd regulations of the Pharmacopoeia of the People's Republic of China.Get preparation antigen and carry out the endotoxin detection.
The preparation of S2.DC tumor vaccine and detection
Gather 3 volunteer peripheral bloods, the peripheral blood that adopts artificial anticoagulant heparin to gather places to be transported to laboratory on ice; 2; 000r/min, 10min, 20 ℃ obtain hemocyte; After diluting 2 times, Ficoll partition method washed corpuscles obtains once fresh PMNC (peripheral blood mononuclear cell PBMC).Be laid in the six porocyte culture plates behind the PBMC with the resuspended prepared fresh of RPMI 1640 culture medium, be positioned over 37 ℃, 5%CO
2Behind the 90min, flush away is attached cell not in the incubator, in culture plate, adds and contains 100ng/mL GM-CSF; RPMI 1640 complete mediums of 50ng/mL IL-4; 3d changes liquid, and 5d gathers in the crops immature DC (imDC), adds apoptotic tumor cell antigen (the antigenic ratio of DC and target cell is 3: 1); To 37 ℃, 5%CO
2Incubator is cultivated 48h jointly, results DC cell, and tongue expects that cell counting is carried out in blue dyeing and phenotype detects.Carry out streaming and detect HLA-DR
+CD11c
+, CD11c
+CD86
+, CD11c
+CD83
+Show the generally acknowledged sign HLA-DR of DC through the flow cytometer facs analysis
+CD11c
+Meansigma methods apoptosis group be 90.90%, the maturation sign CD11c of DC
+CD83
+Meansigma methods apoptosis group be 73.03%, the maturation sign CD11c of DC
+CD86
+Meansigma methods apoptosis group be 86.04%.
This research method adopts and earlier tumor cell is in the heat shock state; Inducing cell high expressed heat shock protein (HSP); Reuse chemicals cell death inducing and then be prepared into the apoptotic cell antigen that is rich in heat shock protein, antigen for preparing and immature DC mixing with cells are cultivated and are obtained sophisticated DC vaccine.The DC vaccine of heat shock brain glioblastoma cell load has apoptosis rate preferably, and through Flow cytometry, the DC vaccine has the phenotypic characteristic of ripe DC, the generally acknowledged sign HLA-DR of DC
+CD11c
+Meansigma methods be 90.90%, the maturation sign CD11c of DC
+CD86
+Meansigma methods be 73.03%, CD11c
+CD83
+Meansigma methods be 86.04%, high expressed CD83, CD86.Show that the DC vaccine Maturity that adopts the load of heat shock method to prepare is high, can be used for induce immune response.
The present invention is not limited to described embodiment, and those skilled in the art still can do some corrections or change, so rights protection scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention promptly openly in the scope.
Claims (1)
1. the method for preparing of a heat shock apoptosis cerebral glioma loaded dendritic cell tumor vaccine, it comprises the steps:
S1. the preparation of heat shock apoptosis brain glioblastoma cell and detection comprises:
The human glioma cell line U251 that adopts draws the ATCC from the U.S., by the conventional method cultivation of going down to posterity; With RPMI1640 culture medium adjustment cell concentration to 10
6/ ml places 40 ℃ with cell, 5%CO
23h in the incubator takes out Tissue Culture Flask, and adding betulinic acid to final concentration is 10 μ g/ml, Tissue Culture Flask is moved to 37 ℃, 5%CO
2Incubator continues to cultivate cell death inducing; The results apoptotic cell adds aseptic PBS washing 4 times, obtains apoptotic tumor cell antigen;
Tumor cell carries out streaming detection computations apoptosis rate result through 40 ℃ after marking cell with FITC-Annexin-V with PI pair after handling 3h;
Soft agarose cloning technique detects the external one-tenth tumor of cellular antigens ability; Get preparation antigen and carry out the endotoxin detection;
The preparation of S2.DC tumor vaccine and detection comprise:
Gather human peripheral blood, the peripheral blood that adopts artificial anticoagulant heparin to gather places to be transported to laboratory on ice; 2,000r/min, 10min; 20 ℃ obtain hemocytees, dilute 2 times after, Ficoll partition method washed corpuscles obtains once fresh PMNC;
Be laid in the six porocyte culture plates behind the PBMC with the resuspended prepared fresh of RPMI 1640 culture medium, be positioned over 37 ℃, 5%CO
2Behind the 90min, flush away is attached cell not in the incubator, in culture plate, adds and contains 100ng/mL GM-CSF; RPMI 1640 complete mediums of 50ng/mL IL-4,3d changes liquid, and 5d gathers in the crops immature DC (imDC); Add apoptotic tumor cell antigen, to 37 ℃, 5%CO
2Incubator is cultivated 48h jointly, results DC cell, and tongue expects that cell counting is carried out in blue dyeing and phenotype detects;
Carry out streaming and detect HLA-DR
+CD11c
+, CD11c
+CD86
+, CD11c
+CD83
+
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104491853A (en) * | 2014-12-29 | 2015-04-08 | 深圳市赛欧细胞生物科技有限公司 | Preparation method of human dendritic cell tumour vaccine |
| CN111334471A (en) * | 2020-03-27 | 2020-06-26 | 郑州鲲鹏健康科技有限公司 | PBMC (peripheral blood mononuclear cell) in-vitro 3D agarose hydrogel culture medium and preparation method thereof |
Citations (2)
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| CN101052709A (en) * | 2004-10-25 | 2007-10-10 | 贝勒研究院 | Dendritic cells loaded with heat shocked melanoma cell bodies |
| CN101918544A (en) * | 2007-12-10 | 2010-12-15 | 因斯布鲁克医科大学 | Method for enhancing immunoreactivity |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052709A (en) * | 2004-10-25 | 2007-10-10 | 贝勒研究院 | Dendritic cells loaded with heat shocked melanoma cell bodies |
| CN101918544A (en) * | 2007-12-10 | 2010-12-15 | 因斯布鲁克医科大学 | Method for enhancing immunoreactivity |
Non-Patent Citations (1)
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| 徐杰等: "热休克诱导凋亡的脑胶质瘤细胞负载树突状细胞及其激发T细胞的体外试验", 《苏州大学学报(医学版)》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104491853A (en) * | 2014-12-29 | 2015-04-08 | 深圳市赛欧细胞生物科技有限公司 | Preparation method of human dendritic cell tumour vaccine |
| CN111334471A (en) * | 2020-03-27 | 2020-06-26 | 郑州鲲鹏健康科技有限公司 | PBMC (peripheral blood mononuclear cell) in-vitro 3D agarose hydrogel culture medium and preparation method thereof |
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Free format text: CORRECT: ADDRESS; FROM: 214000 WUXI, JIANGSU PROVINCE TO: 214043 WUXI, JIANGSU PROVINCE Free format text: CORRECT: INVENTOR; FROM: LU HUA TO: LU HUA CHANG JING |
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| TA01 | Transfer of patent application right |
Effective date of registration: 20130725 Address after: 214043, Jiangsu, Wuxi province Beitang District No. 401 North Hing Yuen Road, north science and Technology Park, room 836 Applicant after: Jiangsu Maijian Bio-tech Development Co.,Ltd. Address before: 214000 Jinshan north science and Technology Industrial Park, No. 888 Jianghai West Road, Jiangsu, Wuxi Applicant before: Lu Hua |
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Application publication date: 20120711 |