CN114574444B - Application of autologous fibroblasts in preparation of anti-rheumatoid arthritis drugs - Google Patents

Abstract

The invention discloses application of autologous fibroblasts in preparing anti-rheumatoid arthritis drugs. The invention provides a recombinant cell which is an in vitro fibroblast containing an expression antirheumatic drug protein coding gene. The antirheumatic protein is DD.E protein or DD.E protein with signal peptide or protein composition with active component DD.E protein. The invention sets up a fibroblast (Dermal Fibroblast cell, DF) based drug delivery platform that can be used for marketing products: antirheumatic drug/fusion protein administration. The invention uses autologous cells to secrete antirheumatic drugs, so that the drugs can be gradually produced in vivo and locally produced, thereby achieving the purpose of treating chronic diseases.

Description

Application of autologous fibroblasts in preparation of anti-rheumatoid arthritis drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of autologous fibroblasts in preparation of anti-rheumatoid arthritis drugs.

Background

Biomacromolecule drugs are globally recognized as the most sophisticated and leading-edge research field in drug research and development in the 21 st century. The development of biomacromolecule drugs represented by protein drugs is particularly rapid. Compared with small molecular chemical medicines, the protein medicine has the characteristics of high specificity, small toxic and side effects, clear action mechanism and high clinical success rate. The development and research fields of macromolecular medicaments adopted for treating tumors by taking tumors as targets are particularly active. Approximately 100 drugs are approved for clinical or preclinical human testing.

In rheumatoid arthritis, tumor necrosis factor- α (TNF- α) is a critical inflammatory cytokine, a factor that is produced locally in large amounts by massive lymphocyte and macrophage infiltration into the synovium of the joint, locally mediating joint damage, while affecting other organs and systems. Tumor necrosis factor antagonists prevent their inflammatory effects by binding to tumor necrosis factor. The medicine based on the tumor necrosis factor (TNF-alpha) inhibitor has definite curative effect in treating rheumatoid arthritis and ankylosing spondylitis, has good tolerance, and can treat autoimmune diseases such as juvenile rheumatoid arthritis, psoriasis and the like. The medicine has quick response for treating rheumatism, and symptoms can be improved within 2-4 weeks, and clinical symptoms can be improved continuously for 3-6 months. Enbrel's drug is also a tumor necrosis factor inhibitor which is a fusion protein of the p75 and IgGFc segments of the human recombinant TNF-alpha receptor. However, it has to be administered continuously, 75mg weekly, with large useful amounts, expensive and systemic disadvantages.

The current methods of administration in clinic are usually in vitro, and are methods of treatment for injection into the human body, which result in very large doses that require treatment, which are very expensive, because systemic administration may cause side effects.

The study of autologous cells to treat tumors is a very exciting field. Such as the use of modified killer T-cell specific tumor cell killing CAR-T (CHIMERIC ANTIGEN receptor) technology, to successfully treat leukemia patients. The first therapeutic vaccine was approved by the FDA in 2010, stimulated in vitro with autologous cells, and returned to the patient as a prostate cancer treatment drug Sipuleucel-T. Cell therapy has also made significant progress in the study of hemophilia, immunodeficiency. Cell therapy has become increasingly widely used in clinic.

The existing use of stem cells as drug carriers is attracting extensive interest, but it is unable to provide a large number of cells, whereas fibroblasts are available in large quantities, are cells at the differentiation end, and are not subdivided into different cells. Fibroblasts or fibroblasts are capable of synthesizing and secreting collagen, elastin, and proteoglycans, which form collagen fibers, elastin fibers, reticulum fibers, and matrix components. They play an important role in wound repair. The fibroblast cells have longer survival time in vivo, are easy to have high gene transduction efficiency and are easy to express recombinant proteins.

Disclosure of Invention

It is an object of the present invention to provide a recombinant cell.

The recombinant cell provided by the invention is an in vitro fibroblast cell for expressing antirheumatic drug protein or a coding gene thereof.

In the recombinant cell, the antirheumatic drug protein is DD.E protein or DD.E protein with signal peptide or a protein composition with DD.E protein as active ingredient.

In the above recombinant cells, in the examples of the present invention, DD.E protein is exemplified as a human protein; the amino acid sequence of the DD.E protein is the 20 th to 486 th positions of the sequence 2 in the sequence table; taking DD.E protein with signal peptide as an example, wherein the signal peptide selects IgG heavy chain signal peptide, and the amino acid sequence of the DD.E protein with signal peptide is sequence 2 in a sequence table.

The isolated fibroblasts are autologous cells of a human or animal.

It is another object of the present invention to provide a method for preparing the recombinant cells described above.

The method provided by the invention comprises the following steps: the isolated fibroblast expresses the gene encoding the antirheumatic drug protein to obtain the recombinant cell.

In the method, the expression of the antirheumatic drug coding gene by the isolated fibroblast is that the antirheumatic drug protein coding gene is introduced into the isolated fibroblast;

and/or, the antirheumatic drug protein coding gene is introduced into the isolated fibroblast cells through a lentiviral expression system.

The antirheumatic drug protein coding gene is introduced into the isolated fibroblast through a lentiviral expression system, and is specifically as follows:

1) Constructing a recombinant plasmid pLVX-puro-DD.E;

2) Introducing plasmids pLVX-puro-DD.E, pCMV-dR8.91 and pCMV-VSV-GpMD2.G into LENTI-X293T cells, and packaging to obtain a lentivirus expressing DD.E;

3) And infecting the in vitro fibroblast cells with the DD.E-expressing lentivirus to obtain recombinant cells.

Use of the recombinant cells described above or a culture supernatant thereof in at least one of:

1) Preparing a product for treating or preventing rheumatoid diseases of human or animals;

2) Preparing a product for weakening or inhibiting inflammatory toxic action of TNF-alpha on cells; wherein the cell is a cell expressing a TNF- α receptor;

3) The application of the method in preparing an animal model for screening medicaments for treating or preventing human or animal rheumatoid diseases;

4) Preparing a product of a neutralizing inflammatory substance TNF-alpha;

5) A product for the treatment or prophylaxis of a disease associated with the inflammatory substance TNF- α.

It is a further object of the present invention to provide a functional product having at least one of the following 1) -4).

The invention provides a product comprising the recombinant cell described above or a culture supernatant thereof;

1) A product for the treatment or prophylaxis of rheumatoid diseases in humans or animals;

2) Products that attenuate or inhibit the inflammatory toxic effects of TNF- α on cells; wherein the cell is a cell expressing a TNF- α receptor;

3) Neutralizing the inflammatory substance TNF- α;

4) Treating or preventing diseases associated with the inflammatory substance TNF-alpha.

It is a further object of the present invention to provide a protein drug delivery system for the treatment or prevention of rheumatoid diseases in humans or animals.

The present invention provides a protein drug delivery system comprising the recombinant cell of the first object.

The use of autologous fibroblasts in the preparation of protein drug delivery systems is also within the scope of the present invention;

Or, the use of autologous fibroblasts and protein drugs in the preparation of protein drug delivery systems is also within the scope of the present invention;

alternatively, the use of autologous fibroblasts and anti-rheumatoid protein drugs for the preparation of protein drug delivery systems for the treatment or prevention of rheumatoid diseases in humans or animals is also within the scope of the present invention.

The invention sets up a fibroblast (Dermal Fibroblast cell, DF) based drug delivery platform that can be used for marketing products: antirheumatic drug/fusion protein administration. The invention uses autologous cells to secrete biomacromolecule/macromolecular drugs, so that the drugs can be gradually produced in vivo and locally produced, thereby achieving the purpose of treating chronic diseases.

Drawings

FIG. 1 is a schematic diagram of the structure of pLVX-pruo plasmid and control plasmid pLVX-EF 1. Alpha. -IRES-ZsGreen 1.

FIG. 2 is a schematic diagram of the structure of DD.E protein.

FIG. 3 shows that DD.E is stably expressed in RDF (RAT DERMAL fibre blank cells) cells.

FIG. 4 shows that RDF cells (RDF/DD.E) transformed with the drug gene DD.E can continuously express DD.E protein.

FIG. 5 shows DD.E neutralization of TNF- α experiments.

FIG. 6 shows DD.E gene transformed RDF cells (RDF/DD.E) expressing DD.E protein and its growth in animals.

Fig. 7 is a custom arthritis grade.

FIG. 8 is a photograph of the hind leg and heel of a rat after induction of rheumatoid arthritis before and after treatment with RDF/DD.E.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Some reagents in the following examples were as follows:

D10 medium: DMEM (ThermoFisher Scientific, cat: 11965118), 10% (volume percent) FCS,20mMGlutaMAX-1 (ThermoFisher scientific. Cat: 35050-61) was added.

Penicillin-Streptomycin:10,000units/ML PENICILLIN and 10,000g/mL streptomycin GIBCO.Cat:15-140-122, the balance being normal saline.

3.Puromuycin:ThermoFisher Scientific，Cat:A1113802。

4. Rat dermal fibroblasts passaged cell complete medium (RDF complete medium): medium106 (ThermoFisher Scientific, cat M106500) was added Low serum Growth SupplememntLSGS (ThermoFisherScinetific cat: S-003-10) at the manufacturer' S recommended concentration, penicillin100 units/ml-Streptomycin mg/ml.

5.Opti-MEM：ThermoFisher Scientific，Cat 31985-062)。

6. Etanercept: enli, etanercept for injection, kang Dele big pharmacy.

7. Transduction reagent Lipofectamine ^TM 2000: thermoFisherScientific, cat:11668027.

8. Lentivirus titer rapid detection card: antaiji (Beijing) Biotechnology Co., ltd., cat: ATG-LT.

9.Polybrene:Sigma-Aldrich,Cat.TR1003。

10.0.45Um filter: pioneering is created, cat: SLHP033RB.

LENTI-X293T cells: clontech, cat, 632180.

Sd rat fibroblasts (RAT DERMAL fibribollast cells, RDF): taking the outer 2mm X2 mm of the SD rat ear with scissors; the fragments were mounted on a cell culture dish by shearing with scissors and incubated at 37℃for 1 hour. The fibroblasts gradually climbed out of the skin tissue with careful injection of 10ml of RDF complete medium. Passaging, the cells can be passaged for more than 20 generations.

13. Immunodeficient mice BALB/c-nu: beijing Vitolihua laboratory animal technology Co., 4 week old rats;

cd (SD) rat: beijing Vitolihua laboratory animal technology Co., ltd; 4-6 weeks old.

Plasmids required for the lentiviral system in the examples below:

pLVX-pruo (DNA sequence shown in FIG. 1, cat: LM1465, shanghai-associated bioengineering Co., ltd.);

pCMV-dR8.91 (Shanghai-Mei bioengineering Co., ltd., LM-1441);

pCMV-VSV-GpMD2.G (WUHan vast, biotechnology Co., ltd., cat: VSV-G);

4. Control plasmid pLVX-EF1 alpha-IRES-ZsGreen 1 (cat: YB2014, the DNA sequence of which is shown in FIG. 1, shanghai Yubo Biotechnology Co., ltd.).

The ELISA method in the following examples was as follows:

1. Reagents and materials

1. Anti-human IgG Sigma-ALDRICH CAT:I3382 mg/ml;

2. anti-human IgG-biotin, sigma-ALDRICH CAT, SAB3701279;

Avidin-HRP 18-4100-51, shanghai Yao rhyme Biotechnology Co., ltd;

4.NuncMaxsorp:thermosCat:44-2404-21；

5.TMB:Solarbio Cat:PR1210；

PBS-T: tween 20.05% in PBS;

7. sealing liquid: 2.5% skim milk powder was dissolved in PBS without Tween20;

8. Antibody/sample dilution: d10 medium DMEM (ThermoFisher Scientific, cat: 11965118) supplemented with 10% (v/v) FCS;

9. standard substance: etanercept (Enli, etanercept for injection Kang Dele big pharmacy)

2. The method comprises the following steps:

1. Coating: anti-human IgG was diluted with PB buffer 3 ug/ml. 50ul of the reaction wells of each ELISA plate was added at 37℃for 2 hours or 4℃overnight. The solution in the wells was discarded, and washed 3 times with washing buffer (PBS-T) (abbreviated as washing, hereinafter referred to as "washing").

2. Closing: 200ul of blocking solution was added to each well and blocked for 1 hour at 37 degrees (no washing after blocking).

3. Sample adding:

Standard preparation: the etanercept standard was comparably diluted with D10 medium to concentrations of 400, 200, 100, 50, 25, 12.5,6.25ng/ml, respectively.

Sample preparation by diluting the sample with the sample diluent D10 in a respective fold ratio, the cell culture supernatant being diluted 20-fold or more, depending on the concentration the sample may express.

Sample adding: the blocking solution is discarded, 0.05ml of diluted sample to be detected is added into the coated reaction holes, and the sample diluent is added into each row of first holes. Incubate at 37℃for 1 hour. Then washed 3 times with the washing solution.

4. Anti-human IgG-biotin was added at 1:1000 fold dilution. Incubate at 37℃for 1 hour and wash 3 times.

5. Avidin-HRP was added at 1:5000 dilution. Incubate at 37℃for 0.5h and wash 3 times.

6. TMB substrate solution was added to develop color, 0.1ml per well, and reacted at room temperature until blue color appeared.

7. Terminating the reaction: to each reaction well was added 50ul of 0.5M sulfuric acid. OD was measured at 450 nm.

EXAMPLE 1 preparation of autologous fibroblast protein drug delivery System

1. DD.E gene with signal peptide

DD.E is a fusion protein consisting of the p75 segment of the TNF-alpha receptor and the IgGFc segment of human origin, and the amino acid sequence of the fusion protein is the same as that of the commercially available drug etanercept (external administration). Commercially available etanercept is a recombinant protein derived from in vitro cell culture and purified from cell culture supernatants.

The invention needs to express DD.E in the transgenic fibroblast, and the transgenic cell is injected into human body, and the cell can continuously synthesize in human body and secrete DD.E out of the cell, thus playing a role of continuous in vivo administration. In order to allow secretion of the synthesized DD.E protein outside the cell, a gene encoding a signal peptide is added before the DD.E gene, the signal peptide is not present in the mature DD.E protein and is decomposed during secretion of the protein.

The nucleotide sequence of DD.E gene with signal peptide is sequence 1 in the sequence table, the 11 th-67 th nucleotide sequence is coding signal peptide sequence, the 68 th-772 th nucleotide sequence is coding human TNF-alpha receptor p75 sequence, the 773 th-1471 th nucleotide sequence is coding human IgGFc segment sequence.

The DD.E gene coded protein with signal peptide is DD.E protein (human source) with signal peptide, its amino acid sequence is sequence 2 in sequence table, in sequence 2 the 1 st-19 th position is signal peptide, the 20 th-254 th position is TNF-alpha receptor p75 section, 255 th-486 th position is IgGFc section; the mature protein is DD.E protein (20 th to 486 th positions of sequence 2).

2. Preparation of recombinant vector (pLVX-puro-DD.E)

Recombinant vector pLVX-puro-DD.E was obtained by inserting the DD.E gene with signal peptide shown in sequence 1 between EcoR1 and BamH1 cleavage sites of plasmid pLVX-puro to obtain a vector expressing DD.E protein with signal peptide.

The DD.E protein structure with signal peptide is shown in figure 2, A is DD.E protein monomer is fusion protein of TNF-alpha receptor p75 and IgGFc, B is predicted dimer structure of DD.E protein-TNF-alpha receptor p75-IgGFc fusion protein, and the mature protein contains no signal peptide.

3. Lentivirus package

The recombinant vector pLVX-puro-DD.E prepared in the above 2 and the control plasmid pLVX-EF 1. Alpha. -IRES-ZsGreen1 (encoding GFP protein) require the use of lentiviruses as vectors to bring the desired gene or control gene into fibroblasts. The specific method comprises the following steps:

1) Packaging lentiviruses

The first day: LENTI-X293T cells were harvested, cell concentrations were adjusted to 4X10 ⁵ cells/1 ml with D10 medium, and 2ml cells were added to 2 wells of a 6-well dish, each well corresponding to 8X10 ⁵ cells.

The following day: cell culture supernatants were carefully aspirated from the edge of the 6-well plate overnight in the morning with a pipette. The D10 medium without Penicillin-Streptomycin was added. Care is taken not to touch the cells.

Preparation of virus by transfecting cells in the afternoon: 100ul of Opti-MEM was added to each of the two EP tubes, 1.5ug of recombinant vector pLVX-puro-DD.E expressing the target gene was added to one EP tube, 1.5ug of control plasmid pLVX-EF 1. Alpha. -IRES-ZsGreen1 was added to the other EP tube, 1 ug of pCMV-dR8.91 (packaging viral plasmid encoding gag-pol-rev protein) and 0.8 ug of plasmid pCMV-VSV-GpMD2.G (packaging plasmid encoding VSV-G protein) were added to each EP tube, and finally 48 ul of Lipofectamine ^TM were added, mixed and left at room temperature for 20 minutes to obtain a plasmid mixture.

Mu.l of each of the above two plasmid mixtures was added dropwise to the above cells cultured overnight, shaking was carried out while adding, and the mixture was incubated overnight at 37 ℃.

Third day: in the morning, the cell culture supernatant on the 6-well dish was carefully aspirated with a pipette and replaced with 2ml of D10 medium containing Penicillin (100 units/ml) -Streptomycin (100. Mu.g/ml). Incubation was continued at 37 ℃.

Fourth day: collecting culture supernatant, and removing cells with a filter with a pore diameter of 0.45 μm to obtain filtrate, namely lentiviral suspension containing DD.E gene with signal peptide and lentiviral suspension containing control Gene (GFP).

2) Lentivirus detection

And (3) detecting the lentivirus suspension containing the DD.E gene with the signal peptide and the lentivirus suspension containing the control Gene (GFP) by using a lentivirus titer rapid detection card, and judging the virus packaging effect.

The existence of a specific detection strip on the detection card and the shade of the color of the strip are used as standards for judging the successful packaging of viruses.

The results were all packed successfully.

4. Preparation of RDF/DD.E cells expressing DD.E protein and cells expressing control RDF/GFP by lentiviral transduced cells

The target gene is integrated into the cell through a viral vector, and the specific steps are as follows:

The first day: the lentivirus transduction was preceded by 18-24 hours, RDF (rat fibroblasts) 0.35X10. 10 ⁶ cells were suspended in 2ml RDF complete medium, seeded into a well of a 6-well cell culture dish, and cultured at 37 ℃.

The following day: 1mlRDF of complete medium in the cell well was carefully removed without touching the cells, 1mL of the above 3 was added to prepare a lentiviral suspension containing DD.E gene with signal peptide and a lentiviral suspension containing control Gene (GFP) (MOI values were about 10), polybrene was added to 8. Mu.g/mL, and to increase transduction efficiency, the cells were centrifuged at 32℃for 180 g for 90 minutes, and then incubated at 37℃for 2 hours, and fresh RDF complete medium was changed to continue culturing overnight. The next day 1. Mu.g/mL puromycin of the wells containing lentiviral suspension with DD.E gene with signal peptide were added to screen puromycin resistant cells. Culturing for 1 week to obtain RDF/DD.E cells expressing DD.E protein; and the lentiviral suspension hole containing the control Gene (GFP) is correspondingly separated from the cells expressing the control protein GFP by a cell flow instrument to obtain RDF/GFP control cells expressing GFP.

5. Identification of DD.E-secreting drugs by fibroblasts expressing DD.E protein (RDF/DD.E)

Whether the structure of the protein expressed by RDF/DD.E cells is consistent with the design, whether the protein can be secreted outside the cells, whether the protein can be folded into a certain conformation in vitro, and whether the protein can neutralize the inflammatory substance TNF-alpha. How the drug is metabolized in the animal and whether it can treat rheumatoid arthritis in the animal, these all need to be tested to verify, specifically as follows:

1) Western Blotting detection of whether RDF/DD.E cells are capable of expressing recombinant DD.E protein

The RDF/DD.E cells expressing DD.E protein and the RDF/GFP cells expressing control protein GFP obtained in the above 4 were cultured overnight in vitro with RDF complete medium, respectively, and the cell supernatants were collected overnight.

Reduction heat treatment: the respective culture supernatants were mixed with 2 mercaptoethanol (reduced RD) and heated at 96℃for 10 minutes to obtain culture supernatants after the reduction heat treatment, and Western Blotting analysis was performed.

Non-reducing NR: the respective culture supernatants were directly subjected to Western Blotting analysis.

The antibody is anti-human IgG.

As shown in FIG. 3, lane 1 from left to right, excluding markers, is the RDF/GFP cell culture supernatant after the reduction heat treatment; lane 2 is the RDF/DD.E cell culture supernatant after the reduction heat treatment; lane 3 is RDF/DD.E cell culture supernatant; as can be seen, the protein detected in lane 2 is around 75kD, which corresponds to the designed protein size; lane 3 detects proteins of 140kD or more in size, since the disulfide bonds of the protein remain intact and fuse the protein dimers without treatment with a reducing agent and without heating. Lane 1 did not detect western blots of designed protein size at 75 kD. It was shown that DD.E protein was about 75kD in the reduced state and about 150kD in the non-reduced state, indicating that it was a dimer formed from two monomers.

2) ELISA method for detecting whether DD.E is continuously expressed in RDF/DD.E cells

The RDF/DD.E cells expressing DD.E protein obtained in the above 4 were cultured in RDF passage cell complete medium, and cell culture supernatant was continuously collected for 50 days. The concentration of the mature DD.E protein in the cell culture supernatant was quantitatively determined by ELISA.

As a result, as shown in FIG. 4, DD.E protein was still expressed in the supernatant of the cells cultured in vitro on day 45, and the expression level was not significantly decreased during the period of 50 days of the culture. The expression of the mature DD.E protein in RDF/DD.E cells is not obviously reduced in the detected time period, and the stable expression can be continued.

The above test results show that RDF/DD.E cells can be used as a rat autologous fibroblast protein drug delivery system.

3) In vitro neutralization assay of inflammatory substances TNF-alpha

The materials are as follows:

a) L929 cells: gmbH, living technologies Co., ltd., cat: CL-0137, expressing the TNF-alpha receptor;

b) RPMI1640 (ThermoFisher Cat: A4192301) was supplemented with 10% (v/v) FCS;

c) Analysis Medium R2 RPMI1640 (ThermoFisher Cat: A4192301) was supplemented with 2% (v/v) FCS;

d) 96-well flat bottom plate: costar, cat:3595;

e) Actinomycin D (Actinomycin D): beijing cool pacing technology Co., ltd., cat:50-76-0CA1201-2mg. 500. Mu.g/mL of the stock solution was stored at-80℃in the dark.

F) CCK8 kit: shanghai assist, saint Biotech Co., ltd., cat: CCK8;

g)TNF-α:Peprotec,Cat:300-01A

the method comprises the following steps:

a) L929 cells were harvested, the cells were suspended in medium R10, and the cells were adjusted to 3.5X10 ⁵/ml. On a 96-well cell culture dish, 100. Mu.l L929 cells were added to each well and cultured overnight at 37 ℃.

B) A mixture of test RDF/dd.e cell culture supernatants and negative control RDF/GFP cell culture supernatants, respectively, was prepared with inflammatory factor TNF- α:

The RDF/DD.E cells expressing DD.E protein and the RDF/GFP cells expressing control protein GFP obtained in the above 4 were cultured overnight in vitro with RDF complete medium, respectively, and the cell supernatants were collected overnight.

The RDF/DD.E culture supernatant or the negative control sample RDF/GFP cell culture supernatant was serially diluted 2-fold with the assay medium R2. Then 100. Mu.l of the above dilution was taken into a blank 96-well cell culture dish, while positive blank control wells were set, to which 100. Mu.l of assay medium R2 was added. To all of the above wells, 100. Mu.l of TNF-. Alpha.solution at a concentration of 2ng/ml (diluted with assay medium R2) was added, respectively. Mixing well, incubating at 37 ℃ for 2 hours to obtain a mixture.

C) Cell supernatants of overnight cultures of a) L929 cells were discarded, and 50. Mu.l of the mixture obtained in b) was transferred to wells containing L929 cells from which the supernatant was discarded, respectively. Then 50. Mu.l of actinomycin D (diluted to 4. Mu.g/mL with the medium for analysis R2) was added to each well. Culturing at 37 ℃ for 24 hours.

D) 10% CCK8 solution was added directly to each well. The reagent contains chemical components which can be reduced by dehydrogenase in living cells to yellow formazan product with high water solubility, and the amount of the chemical components is proportional to the number of the living cells. Thus, this property can be used to directly conduct cell proliferation and toxicity analysis. Incubation is carried out for 1-4 hours. The absorbance was read by a microplate reader. OD ₄₅₀ was measured 2 hours after addition of CCK8 at a wavelength of 450 nm.

E) The OD ₁₅₀ value of the positive control wells was calculated as 100% viable, while the ratio of OD ₄₅₀ values measured for other cells per well to OD ₄₅₀ of the positive control wells was used to make a "neutralization curve", the higher the ratio, the more components in the cell culture supernatant that were able to neutralize TNF- α toxicity, the less toxic effect TNF- α has on the cells and the more protected the cells.

The results are shown in FIG. 5, two curves, the upper curve being a graph of the effect of neutralizing TNF- α in the experimental RDF/DD.E cell culture supernatant and the lower curve being a set of graphs of the effect of neutralizing TNF- α in the RDF/GFP cell culture supernatant of the negative control sample. Experimental results show that the secretion of DD.E by the culture supernatant of RDF/DD.E cells can weaken or inhibit the toxic effect of TNF-alpha on L929 cells; the toxic effect of the inflammatory factor TNF-alpha on L293 cells could not be neutralized in the negative control RDF/GFP cell culture supernatant; DD.E was shown to neutralize the toxic effects of TNF- α on L929 cells, and such neutralization was reduced with reduced DD.E dose. The attenuation or inhibition of the toxic effects of TNF- α on L929 cells by dd.e. is drug specific.

6. Metabolism of recombinant DD.E protein expressed by fibroblast RDF/DD.E in nude mice and SD rats (autologous)

RDF cells are derived from SD rats, and RDF is cleared by the immune system in normal mice, so that nude mice without immune function are used for the following experiments to examine the condition that RDF/DD.E cells express DD.E protein in nude mice. RDF/DD.E cells were also examined in SD (autologous) rats for expression of DD.E protein.

1) Expression of DD.E protein in immunodeficient mice

Experimental group test mice: 1.6X10 ⁷ of the obtained RDF/DD.E cells expressing DD.E protein obtained in the above 4 were suspended in 0.2ml of physiological saline, and the cell suspensions were injected subcutaneously into 2 immunodeficient mice BALB/c-nu (18 g size) at a dose of 0.1ml of 8X10 ⁶ cells per mouse, respectively; the injection mode was subcutaneous measured in the proximal leg abdomen and designated as laboratory mice 1 and 2.

Control group control mice: 1.6X10 ⁷ of the RDF/GFP cells expressing the control protein GFP obtained in 4 above were suspended in 0.2ml of physiological saline, and the cell suspensions were injected subcutaneously into 2 immunodeficient mice BALB/c-nu (18 g size) at a dose of 0.1ml of 8X10 ⁶ cells per mouse. The injection mode was subcutaneous measured in the proximal leg abdomen and designated control mice 1 and 2.

The blood of the tail of the mouse is collected before injection and 1-22 days after injection, and Western blotting is performed to detect whether DD.E protein exists in the blood of the mouse.

As shown in FIG. 6A, blood is collected from the front and back tails of RDF/DD.E and negative control cells (RDF/GFP) of nude mice for injection experiments, and the Westernbloting method is used for detecting the existence and the growth of DD.E in the blood; this is the test result of one test mouse and one control test mouse. It can be seen that mice started to detect the presence of dd.e in blood the following day after RDF/dd.e cell injection, and that dd.e expression had a tendency to increase gradually over time. On days 15 and 22, dd.e protein could still be detected. The secondary antibody adopted in the test has cross reaction with the mouse IgG, and the cross reaction band is about 45kD, so that the observation of 75kD protein is not affected, and the secondary antibody is used as an ideal internal reference for sample loading.

The body weight and viscera conditions of the mice after injection were checked at the same time, and the body weight results of the mice are shown in Table 1, and it can be seen that the body weight did not change significantly before and after injection, nor did viscera change detected.

Table 1 shows the body weight changes before and after injecting test cells RDF/DD.E and negative control cells (RDF/GFP) into nude mice

Weight of body	Test mouse 1	Test mouse 2	Control mouse 1	Control mouse 2
					dayO	18.1	18.8	18.5	18.4
day1	20.2	19.2	19.1	18.9
					day2	20	19.4	19.2	19.1
day3	19.4	19.4	19.5	19.4
					day4	19.6	19.3	19.4	19.5
day5	18.4	182	19.5	19.4
					day15	0.6	19.4	19.6	19.6
day17	21.1	19.9	19.7	19.5
					day22	21	19.9.	19.7	19.9

2) Expression of DD.E protein in SD rat (autologous)

Experimental group experimental rats: suspending 18x10 ⁷ RDF/DD.E cells expressing DD.E protein obtained in the above 4 in 0.6ml physiological saline, and subcutaneously injecting the cell suspension into the back leg of #3, #4#5 SD rat, wherein the injection dose is 0.2ml of 6x10 ⁷ cells per rat; the injection mode is subcutaneous measurement at the proximal leg abdomen.

Control group control mice: 18X10 ⁷ RDF/GFP cells expressing control protein GFP obtained in 4 above were suspended in 0.6ml of physiological saline, and the cell suspensions were subcutaneously injected into the insides of the hind legs of SD rats #1, #2 and #3, respectively, at a dose of 0.2ml of cells containing 6X10 ⁷ cells per rat. The injection mode is subcutaneous measurement at the proximal leg abdomen.

Rat tail blood was collected before and 1-50 days after injection, serum was isolated, and the DD.E protein content in the serum was measured by ELISA.

As a result, as shown in FIG. 6B, the concentration of DD.E medicine in blood was quantitatively measured by ELISA method from tail blood collection before and after injecting experimental cell RDF/DD.E (mice 3,5, 6) and negative control cell RDF/GFP (mice 2, 4) into SD rat; DD.E has a gradual upward trend in the SD rats of the test group for 1-15 days, and the DD.E is maintained at about 30ng/ml in 16-50 days; while DD.E was negative in control SD rats, no DD.E was detected.

Thus, RDF/DD.E cells expressing DD.E protein can be used as autologous fibroblast protein drug delivery system to achieve sustained expression of mature DD.E protein in rats.

EXAMPLE 2 use of autologous fibroblast protein drug delivery System

1. Preparation of animal arthritis model for SD rats

The arthritis model of the induced rat is a relatively mature technology.

Reagent:

1) Bovine type II collagen: chondrex, cat 20022;

2) Freund's complete adjuvant: sigma-Aldrich, cat: F5881;

The method comprises the following steps:

1) Bovine type II collagen was dissolved in PBS to give a bovine type II collagen solution at a concentration of 2 mg/ml. Taking 0.5ml of bovine type II collagen solution and 0.5ml of Freund's complete adjuvant to obtain injection.

2) Multiple intradermal injections were made at the tail and heel of SD rats. Each rat was injected with 0.1ml of the injection obtained in 1) above, and the hind paw pad of the rat became thicker and red and swollen within 7 to 30 days. 6 SD rats were induced with collagen, and the rats developed successively within 1 month. Murine #1, 4 did not develop disease. The thickness of the murine foot plate was measured with calipers. Normal murine foot plate thickness is 4.5cm. The thickness of the foot plate of the affected mice is 4.6cm or more, the mice with the sizes of 2# and 3# and the mice with the sizes of 5# and 6# are affected, the mice are retreated to generate red swelling, and the hind legs are everted.

According to the degree of redness and swelling of SD rats, the severity was classified into 5 grades, the arthritis grade was as shown in fig. 7, grade 0: normal rear legs; stage I: just visible redness and swelling, stiff joints; stage II: obvious reddening and swelling, widening and thickening of the sole and stiff joints; class III: the root of the rear leg is enlarged, the sole is widened and thickened, and the foot cannot land; grade IV: the heel is enlarged, the sole is widened and thickened, the foot cannot land, and the foot is everted.

SD animals had a relatively uniform weight from 4 weeks to 6 weeks (underage) prior to induction of rheumatoid arthritis, and had a tendency to gain in weight at 380 g (+/-10 g). Animals vary widely in body weight prior to sacrifice and are associated with rheumatoid arthritis severity. Pain is predicted to affect appetite and sleep in animals and thus weight gain in animals. The ratio of spleen weight to body weight was substantially constant between animals. And the viscera of the animal were examined, and no abnormality was found in the viscera of the animal.

The incidence and foot thickness of 6 SD rats, body weight, spleen weight, foot weight before and after treatment are shown in Table 2 and FIG. 7.

Table 2 shows the SD rats before and after treatment

2. Application of autologous fibroblast protein medicine in treating arthritis

The animals with disease are divided into treatment groups and negative control treatment groups:

Treatment group SD rats #3 and #6: each rat was subcutaneously injected with 0.2ml of 4X10 ⁷ of the above 4-derived test RDF/DD.E cells suspended in physiological saline under the swollen instep;

control group SD rats #2#5 each injected subcutaneously on the swollen instep with 0.2ml of cells containing 4X10 ⁷ negative control RDF/GFP suspended in physiological saline.

4 Days after injection, the rats were sacrificed, their weights were weighed, their feet were weighed (cut 1cm from the heel), the thickness of the foot plate was measured with a caliper, and their spleen weights were weighed, and the results are shown in table 2 and fig. 8, a picture of the rear leg and heel of the rat after induction of rheumatoid arthritis; 4 mice developed. The hind legs all reach grade IV. Mice #2, #5 were treated with control and #3, #6, and negative control cells, RDF/GFP, were injected subcutaneously into the back of mice #2 and # 5. Therapeutic cells (RDF/DD.E) were injected subcutaneously into the hind leg backs of mice #3 and # 6. The recovery from murine disease was checked after 4 days of treatment. The leg swelling of the #3 mice and the #6 mice is obviously improved, the hind legs can land, and the detumescence degree is 58% and 120% respectively according to the thickness of the foot plate. The degree of inflammation improves to a grade II degree. The degree of inflammation in mice #2 and #5 was not significantly improved and remained at grade IV. Although the feet have detumescence, the detumescence range is only 7.6% and 33%.

Although the number of test animals is small, and the causes of individual differences in animals are not excluded, there is a trend to show that DD.E modified RDF cells (RDF/DD.E cells) have anti-inflammatory effects.

The hind legs of the sacrificed rats were analyzed by CT scan after 4 days of injection, and the results are shown in table 3, and it can be seen that the bone volume of the ill rats was increased, and whether treatment had no significant effect on the bone volume increase. The surface area of the rear leg bones of the animals suffering from the same disease increases. But whether treated or not has little effect on bone surface area. This indicates that bone injury has been irreversible by the time swelling of the hind leg occurs visually. Therefore, in future large-scale animal experiments, treatment should be performed early in onset.

Table 3 shows CT scan results for small animals

SEQUENCE LISTING

<110> Dada Biotechnology (Beijing) Co., ltd

Application of <120> autologous fibroblasts in preparation of anti-rheumatoid arthritis drugs

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1471

<212> DNA

<213> Artificial sequence

<400> 1

gatcgaattc atggactgga cctggaggat actcttcttg gtggcggccg ccacaggcgc 60

gcactccttg cccgcccagg tggcatttac accctacgcc ccggagcccg ggagcacatg 120

ccggctcaga gaatactatg accagacagc tcagatgtgc tgcagcaagt gctcgccggg 180

ccaacatgca aaagtcttct gtaccaagac ctcggacacc gtgtgtgact cctgtgagga 240

cagcacatac acccagctct ggaactgggt tcccgagtgc ttgagctgtg gctcccgctg 300

tagctctgac caggtggaaa ctcaagcctg cactcgggaa cagaaccgca tctgcacctg 360

caggcccggc tggtactgcg cgctgagcaa gcaggagggg tgccggctgt gcgcgccgct 420

gcgcaagtgc cgcccgggct tcggcgtggc cagaccagga actgaaacat cagacgtggt 480

gtgcaagccc tgtgccccgg ggacgttctc caacacgact tcatccacgg atatttgcag 540

gccccaccag atctgtaacg tggtggccat ccctgggaat gcaagcatgg atgcagtctg 600

cacgtccacg tcccccaccc ggagtatggc cccaggggca gtacacttac cccagccagt 660

gtccacacga tcccaacaca cgcagccaac tccagaaccc agcactgctc caagcacctc 720

cttcctgctc ccaatgggcc ccagcccccc agctgaaggg agcactggcg acgagcccaa 780

atcttgtgac aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc 840

gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga 900

ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta 960

cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag 1020

cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga 1080

gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa 1140

agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat 1200

gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc 1260

cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct 1320

ggactccgac ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca 1380

gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca 1440

gaagagcctc tccctgtccc cgggtaaata g 1471

<210> 2

<211> 486

<212> PRT

<213> Artificial sequence

<400> 2

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu

20 25 30

Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln

35 40 45

Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys

50 55 60

Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr

65 70 75 80

Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg

85 90 95

Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn

100 105 110

Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln

115 120 125

Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe

130 135 140

Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro

145 150 155 160

Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys

165 170 175

Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser

180 185 190

Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro

195 200 205

Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr

210 215 220

Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu

225 230 235 240

Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro

245 250 255

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

260 265 270

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

275 280 285

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

290 295 300

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

305 310 315 320

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

325 330 335

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

340 345 350

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

355 360 365

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

370 375 380

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

385 390 395 400

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

405 410 415

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

420 425 430

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

435 440 445

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

450 455 460

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

465 470 475 480

Ser Leu Ser Pro Gly Lys

485

Claims (7)

1. A recombinant cell which is an isolated fibroblast cell expressing an antirheumatic drug protein or a gene encoding the same;

The antirheumatic drug protein is DD.E protein with signal peptide; the amino acid sequence of the DD.E protein with the signal peptide is SEQ ID No.2 in the sequence table.

2. The recombinant cell of claim 1, wherein: the isolated fibroblasts are autologous cells.

3. A method of making the recombinant cell of claim 1 or 2, comprising the steps of: and (3) enabling the isolated fibroblast to express the gene encoding the antirheumatic drug protein, so as to obtain the recombinant cell.

4. A method according to claim 3, characterized in that: the method comprises the steps that the isolated fibroblast expresses an antirheumatic drug coding gene, and the antirheumatic drug protein coding gene is introduced into the isolated fibroblast;

the antirheumatic drug protein coding gene is introduced into the isolated fibroblasts by a lentiviral expression system.

5. Use of the recombinant cell of claim 1 or 2 in at least one of:

1) Preparing a product for treating or preventing rheumatoid diseases of human or animals;

2) The application of the method in preparing an animal model for screening medicaments for treating or preventing human or animal rheumatoid diseases.

6. A product having at least one of the functions 1) -4) comprising the recombinant cell of claim 1 or 2;

1) Treating or preventing rheumatoid diseases of human or animal;

2) Attenuation or inhibition of toxic effects of TNF- α on cells;

3) Neutralizing the inflammatory substance TNF- α;

4) Treating or preventing diseases associated with the inflammatory substance TNF-alpha.

7. A protein drug delivery system for the treatment or prophylaxis of rheumatoid disease in humans or animals comprising the recombinant cell of claim 1 or 2.