WO2020103651A1 - Use of mesenchymal stem cells in preparation of product for treating rheumatoid arthritis - Google Patents

Abstract

Disclosed is the use of mesenchymal stem cells in the preparation of a product for treating rheumatoid arthritis. The present invention claims the use of mesenchymal stem cells or a cell preparation thereof in the preparation of a drug for treating rheumatoid arthritis. The method for preparing the cell preparation comprises the following steps: using an umbilical cord Wharton-area tissue block as a raw material to prepare umbilical cord mesenchymal stem cells, thereby obtaining the cell preparation, wherein a low-serum medium is used in the preparation process. The cell preparation provided by the present invention is suitable for the cell therapy of rheumatoid arthritis and related diseases thereof. The rheumatoid arthritis-related diseases comprise complications of rheumatoid arthritis and diseases with similar pathogenesis thereto, such as rheumatoid vasculitis, pneumonia, osteoarthritis, rheumatoid cardiopathy, ankylosing spondylitis, gout, oculopathy, nephropathy, urinary system infection, Cushing's syndrome and oral ulcers. The present invention has an application value for treating rheumatoid arthritis and related diseases thereof.

Description

Application of mesenchymal stem cells in preparing products for treating rheumatoid arthritis Technical field

The invention relates to the application of mesenchymal stem cells in preparing products for treating rheumatoid arthritis, in particular to the application of human umbilical cord Wharton interval mesenchymal stem cell preparations in preparing products for treating rheumatoid arthritis.

Background technique

Rheumatoid arthritis (rheumatoid arthritis, RA) is a systemic autoimmune disease characterized by synovitis of joints and erosion and destruction of articular cartilage, with a global incidence rate of about 1%. A sample survey in China The result was about 0.8%; the main pathological manifestations were abnormal immune function, hyperplasia of joint synovium, destruction of articular cartilage and bone tissue. The lesions of the joint mainly manifest as inflammatory cell infiltration, synovial hyperplasia, pannus formation, cartilage and synovial damage, etc .; pannus gradually extends from the synovium at the edge of articular cartilage to the cartilage surface, and finally covers the articular cartilage surface Thereby blocking the contact of chondrocytes with synovial fluid, which in turn affects the cartilage tissue receiving nutrients; in addition, excessive hyperplasia and repeated attacks of the synovium can trigger a violent inflammatory reaction, resulting in the destruction of articular cartilage and bone tissue, and joint dysfunction In severe cases, the patient will eventually become disabled.

With the development of molecular biology, immunopathology, genetic engineering technology and other technologies, the research on the etiology and pathogenesis of RA has been in-depth. So far, the specific pathogenesis of RA is still inconclusive; genetic factors, immune cells, cytokines, etc. may play an important role in the pathogenesis of RA. Since the pathogenesis of RA is not very clear, there is no cure. At present, the treatment is mainly to delay the development of the disease, reduce the degree of morbidity, and mainly improve the quality of life of the patients. In the treatment of RA, drug treatment is the most commonly used method. At present, the drugs used to treat RA are mainly divided into symptomatic anti-rheumatic drugs, disease-modifying anti-rheumatic drugs, glucocorticoids, and botanical drugs. In addition, there are methods such as gene therapy and surgery.

Invention Disclosure

The object of the present invention is to provide new uses of mesenchymal stem cells or cell preparations.

The invention first protects the application of mesenchymal stem cells in the preparation of a medicament for treating rheumatoid arthritis.

In the above application, the mesenchymal stem cells are umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared by using tissue blocks of the umbilical cord Wharton area as raw materials. In the process of preparing umbilical cord mesenchymal stem cells using umbilical cord Wharton tissue blocks as raw materials, low serum medium is used. The umbilical cord is an isolated umbilical cord. The umbilical cord mesenchymal stem cells may be primary umbilical cord mesenchymal stem cells or umbilical cord mesenchymal stem cells after passage. The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, and more specifically umbilical cord mesenchymal stem cells passaged for 3-5 passages .

The invention also protects the use of cell preparations in the preparation of medicaments for the treatment of rheumatoid arthritis.

In the above application, the preparation method of the cell preparation includes the following steps: the umbilical cord mesenchymal stem cells are prepared by using the tissue block of the umbilical cord Wharton area as a raw material, and a low serum medium is used in the preparation process.

The umbilical cord mesenchymal stem cells may be primary umbilical cord mesenchymal stem cells or umbilical cord mesenchymal stem cells after passage.

The preparation method of the primary umbilical cord mesenchymal stem cells sequentially includes the following steps:

(1) Cultivate the tissue block of the umbilical cord Wharton with low serum medium until cells crawl out;

(2) Use low serum medium to cultivate cells;

(3) Collect the cells, digest them with trypsin, and collect the cells, which are the primary umbilical cord mesenchymal cells.

The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, and more specifically umbilical cord mesenchymal stem cells passaged 3-5 . The method of passaging may specifically be: passaging the umbilical cord mesenchymal cells 1: 2, culturing the cells with a low serum medium, collecting the cells, digesting with trypsin, and collecting the cells.

Further, the preparation method of the cell preparation includes the following steps:

(1) Use low serum medium to culture the isolated umbilical cord Wharton tissue block for 7-10 days;

(2) After completing step (1), the tissue block is discarded, and the cells are cultured to 80% confluence using a low serum medium;

(3) After completing step (2), collect the cells, digest them with trypsin, and collect the cells, which are P0 generation umbilical cord mesenchymal cells;

(4) After step (3) is completed, the P0 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to 80% confluence using a low serum medium;

(5) After completing step (4), collect the cells, digest them with trypsin, and collect the cells, which are P1 generation umbilical cord mesenchymal cells;

(6) After completing step (5), the P1 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to a 80% confluence using a low serum medium;

(7) After completing step (6), collect the cells, digest with trypsin, and collect the cells, which are P2 generation umbilical cord mesenchymal cells;

(8) After completing step (7), the P2 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to 80% confluence using a low serum medium;

(9) After completing step (8), collect the cells, digest with trypsin, and collect the cells, which is the cell preparation.

Any one of the above cell preparations is a CD126 positive cell preparation, and the CD126 positive rate is above 95%, preferably above 99%. Any one of the above cell preparations is a cell preparation with a positive rate of CD29, CD44, CD90, and CD105 above 90%. The positive rate of CD29, CD44, CD90, and CD105 is preferably above 95%, and more preferably above 99%. The positive rate of CD31 and CD34 of any of the above cell preparations is less than 10%, preferably less than 7%. The CD45 and HLA-DR positive rates of any of the above cell preparations are less than 5%, preferably less than 3%, and more preferably less than 1%. CD126, CD29, CD44, CD90 and CD105 are commonly used surface markers for identifying mesenchymal stem cells. The high positive rate of CD126, CD29, CD44, CD90 and CD105 indicates that the cell preparation is mesenchymal stem cells and their purity is very high. CD31 is a marker of endothelial progenitor cells, CD45 is a marker of white blood cells and CD34 is a marker of hematopoietic stem cells. The positive rates of the three indicators of CD31, CD45 and CD34 are low, indicating that the purity of the cell preparation is very high. HLA-DR is a marker related to transplant rejection. The low HLA-DR positive rate indicates that transplant rejection is not likely to occur when the cell preparation is applied.

The invention also protects a medicine for treating rheumatoid arthritis.

The active ingredient of the medicament for treating rheumatoid arthritis protected by the present invention is mesenchymal stem cells or the above cell preparations.

In the above medicine, the mesenchymal stem cells are umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared by using tissue blocks of the umbilical cord Wharton area as raw materials. The preparation of umbilical cord mesenchymal stem cells using umbilical cord Wharton tissue as raw materials uses low serum medium. The umbilical cord is an isolated umbilical cord. The umbilical cord mesenchymal stem cells may be primary umbilical cord mesenchymal stem cells or umbilical cord mesenchymal stem cells after passage. The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, more specifically 3-5 generations of umbilical cord mesenchymal stem cells.

The low-serum culture medium described above is a cell culture medium containing 3-5% (volume percentage) serum.

The composition of any of the above low-serum culture media is as follows: human epithelial growth factor 10 ng / mL, human basic fibroblast growth factor 10 ng / mL, recombinant human insulin-like growth factor 5 μg / mL, platelet-derived factor 10 ng / mL, heparin 5μg / mL, hydrocortisone 1μg / mL, ascorbic acid 10μg / mL, non-essential amino acid solution 1% (volume percentage), L-glutamine 2mmol / L, fetal bovine serum 4% (volume percentage), penicillin 50U / mL, streptomycin 50μg / mL, the balance is DMEM high glucose medium.

The non-essential amino acid solution contains Glycine 10mM, L-Alanine 10mM, L-Asparagine 10mM, L-Aspartic acid 10mM, L-Glutamic Acid 10mM, L-Proline 10mM, L-Serine 10mM.

In the present invention, low serum medium is used for cultivation. In the culture of mesenchymal stem cells, 10% (volume percentage) fetal bovine serum is usually used, and some have a serum concentration as high as 20% (volume percentage). The serum contains various plasma proteins, peptides, carbohydrates, growth factors, hormones, etc. The composition of serum is complex, and there are differences between each batch of serum. In addition, although the serum contains many components that are conducive to cell growth, it inevitably contains some components that are harmful to the cells, such as complement, antibodies, and endotoxin. Therefore, cells cultured from high-concentration serum are not suitable for clinical application and will increase the risk of clinical allergies. The inventors of the present invention have found that a low serum medium with a serum concentration of 3-5% (volume percentage) has no adverse effects on cell growth, proliferation, morphology and function.

Mesenchymal stem cells commonly used in immunotherapy include bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, fat-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, etc. Compared with commonly used bone marrow mesenchymal stem cells (MSCs), umbilical cord Wharton mesenchymal stem cells have rich sources, no impact on donors, easy collection and transportation, low possibility of carcinogenicity, low probability of virus contamination, and immunogenicity Weakness, no social, ethical and legal disputes and many other advantages. More importantly, the mesenchymal stem cells isolated from the Wharton area of the umbilical cord have high content, higher proliferative capacity than bone marrow MSCs, and lower immunogenicity than bone marrow MSCs.

Cellular therapy is to directly affect the patient's immune system and adjust the patient's immune balance from the perspective of cellular and humoral immunity, thereby achieving the purpose of treating rheumatoid arthritis.

The cell preparation provided by the present invention is suitable for cell therapy of rheumatoid arthritis and related diseases. The rheumatoid arthritis-related diseases include complications of rheumatoid arthritis and diseases with similar pathogenesis, such as rheumatoid vasculitis, pneumonia, osteoarthritis, rheumatoid heart disease, ankylosing spondylitis , Gout, eye disease, kidney disease, urinary system infection, Cushing's syndrome, oral ulcers, etc.

The invention finally protects a method for treating rheumatoid arthritis or related diseases.

The method for treating rheumatoid arthritis or related diseases protected by the present invention includes the following steps: administering the above-mentioned drugs to patients with rheumatoid arthritis or patients with rheumatoid arthritis-related diseases, so that the patients are treated.

In the above method, the rheumatoid arthritis-related diseases include complications of rheumatoid arthritis and diseases with similar pathogenesis, such as the complications of rheumatoid arthritis and diseases with similar pathogenesis include Rheumatic vasculitis, pneumonia, osteoarthritis, rheumatoid heart disease, ankylosing spondylitis, gout, eye disease, kidney disease, urinary tract infections, Cushing's syndrome, and oral ulcers.

BRIEF DESCRIPTION

Figure 1 shows the results of mesenchymal stem cell markers (CD29, CD44, CD90, CD105 and CD126).

Figure 2 shows the results of hematopoietic stem cells and endothelial cell markers (CD31, CD45 and CD34).

Figure 3 shows the results of transplant rejection-related markers (HLA-DR).

Figure 4 shows the results of phenotype observation of arthritis mice.

Figure 5 shows the results of pathological examination of mouse joints.

Figure 6 shows the detection results of mouse serum inflammatory factors IL-17, TNF-α, IL-6 and IFN-γ.

The best way to implement the invention

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The formulation or source of each reagent used in the following examples:

Low serum medium: human epithelial growth factor 10ng / mL, human basic fibroblast growth factor 10ng / mL, recombinant human insulin-like growth factor 5μg / mL, platelet-derived factor 10ng / mL, heparin 5μg / mL, hydrocortisone 1μg / mL, ascorbic acid 10μg / mL, non-essential amino acid solution 1% (volume percentage), L-glutamine 2mmol / L, fetal bovine serum 4% (volume percentage), penicillin 50U / mL, streptomycin 50μg / mL , The balance is DMEM high glucose medium.

Human epithelial growth factor (hEGF): GenScript company, the full name of the product is EGF Recombinant Human Protein, the article number is Z00333.

Human basic fibroblast growth factor (b-FGF): GenScript Corporation, article number Z03116.

Recombinant Human Insulin-like Growth Factor (IGF): GenScript Corporation, Catalog No. Z03017.

Platelet-derived factor (PDGF): GenScript Corporation, article number Z02529.

Chicken type II collagen: Chondrex company, article number 20012.

Freund's complete adjuvant: Chondrex, catalog number 7001.

Freund's incomplete adjuvant: Chondrex, catalog number 7002.

Heparin: Maichen Company.

Hydrocortisone: Maichen Company.

Ascorbic acid: Maichen Company.

Penicillin: GIBCO Corporation.

Streptomycin: GIBCO Corporation.

The non-essential amino acid solution is Gibco ^™ MEM Non-Essential Amino Acids Solution, (100X), article number 11140050. The website is as follows: https://www.thermofisher.com/cn/zh/home/technical-resources/media-formulation.165.html. There are seven kinds of amino acids in this product, the composition is as follows: Glycine 10.0mM, L-Alanine 10.0mM, L-Asparagine 10.0mM, L-Aspartic acid 10.0mM, L-Glutamic Acid 10.0mM, L-Proline 10.0mM, L-Serine 10.0mM.

DBA / 1J mouse: Beijing Viton Lihua Co., Ltd.

Example 1. Preparation of umbilical cord Wharton interval mesenchymal stem cell preparation

1. Obtaining fetal umbilical cord

Take the isolated umbilical cord of the neonate with full-term and no congenital diseases; the mother has no infectious diseases such as hepatitis, syphilis, AIDS, etc. The mother and her family have informed consent to the umbilical cord for experimental research.

2. Obtaining umbilical cord Wharton interval mesenchymal cells

1. Pretreatment of umbilical cord

In a sterile laboratory table, the umbilical cord was repeatedly rinsed with physiological saline to wash away residual blood. With sterile surgical instrument of 2-3cm umbilical cord cut into small pieces, longitudinally cut the umbilical cord, umbilical artery is removed, and the umbilical vein amnion, taking zone Wharton, cut into small pieces of about 0.5-1mm ^3.

2. Acquisition of primary umbilical cord mesenchymal cells

To obtain primary umbilical cord mesenchymal cells using tissue block culture method, the specific steps are as follows:

(1) Spread the tissue block of the Wharton area obtained in step 1 evenly in a sterile petri dish with a diameter of 10 cm, covering 60-70% of the bottom area of the dish, and put it upside down in a 37 ° C incubator for 15 min. Gently add 10 mL of low-serum medium, and incubate at 37 ° C in a 5% CO ₂ incubator for 7-10 days. At this time, cells are crawled out uniformly under the tissue block.

(2) After completing step (1), take the petri dish, wash it twice with PBS buffer, discard the tissue block (at this time the cells have adhered to the growth), add 10mL of fresh low serum medium (3-4 Change the solution once a day), and culture until the cells are about 80% of the bottom of the culture dish.

(3) After completing step (2), collect the cells, digest with 0.25% trypsin for 1-2 min, centrifuge at 1000 rpm for 3 min, collect the cells, which are primary umbilical cord mesenchymal cells, also known as P0 generation umbilical cord mesenchymal cells .

3. Acquisition of P3 generation umbilical cord mesenchymal cells

(1) Divide the P0 generation umbilical cord mesenchymal cells obtained in step 2 into two sterile culture dishes with a diameter of 10 cm (1: 2 passage), and add 10 mL of fresh low serum medium to each culture dish (Change the medium once every 3-4 days), and cultivate until the cells are about 80% of the bottom of the culture dish.

(2) After completing step (1), collect the cells, digest with 0.25% trypsin for 1-2 min, centrifuge at 1000 rpm for 3 min, and collect the cells, which are P1 generation umbilical cord mesenchymal cells.

(3) After completing step (2), the P1 generation umbilical cord mesenchymal cells were evenly divided into sterile culture dishes with a diameter of 10 cm (1: 2 passage), and 10 mL of fresh low serum was added to each culture dish for culture Base (change the medium once every 3-4 days), and culture until the cells cover about 80% of the bottom of the culture dish.

(4) After completing step (3), collect the cells, digest the cells with 0.25% trypsin for 1-2 min, centrifuge at 1000 rpm for 3 min, and collect the cells, which are P2 generation umbilical cord mesenchymal cells.

(5) After completing step (4), divide the P2 generation umbilical cord mesenchymal cells into a sterile culture dish with a diameter of 10cm (1: 2 passage), and add 10mL of fresh low serum culture to each culture dish Base (change the medium once every 3-4 days), and culture until the cells cover about 80% of the bottom of the culture dish.

(6) After completing step (5), collect the cells, digest the cells with 0.25% trypsin for 1-2 min, centrifuge at 1000 rpm for 3 min, and collect the cells, which are P3 generation umbilical cord mesenchymal cells (also called P3 cell preparation).

Example 2. Identification of P3 cell preparation

The test article was the P3 cell preparation prepared in Example 1.

Use flow cytometry to detect the expression of CD29, CD44, CD90, CD105, CD126, CD31, CD45, CD34 and HLA-DR in the test products, the specific steps are as follows: after incubating the cells (test samples) with the corresponding antibodies respectively , Wash off the excess antibody, resuspend the cells in PBS buffer, and use BD's LSR Fortessa instrument to detect the positive rate of each index.

The detection results of the mesenchymal stem cell markers (CD29, CD44, CD90, CD105 and CD126) are shown in Figure 1. The detection results of hematopoietic stem cell markers (CD31, CD45 and CD34) are shown in Figure 2. The test results of transplant rejection-related markers (HLA-DR) are shown in Figure 3. The results showed that the test products highly expressed the markers of mesenchymal stem cells (positive rates were all above 97%), and the low expression of hematopoietic stem cells and endothelial cell markers (positive rates were both 5% lower), basically did not express transplant rejection-related Markers (positive rate is 0.1%).

Example 3. The therapeutic effect of P3 cell preparation on rheumatoid arthritis

1. Preparation of the model and administration

Test mice: 6-8 weeks, DBA / 1J mice, adapted for one week (clean animal feeding room, free intake of drinking water). Mice were sensitized and induced arthritis model with chicken type II collagen and Freund's adjuvant. According to whether it is administered or not, it is divided into the following groups:

Normal control group (5 mice): Each mouse was subcutaneously injected with 0.1 mL of normal saline at the base of the tail. After 3 weeks, each mouse was subcutaneously injected with 0.1 mL of normal saline at the base of the tail. From the day of the second injection, normal saline was injected into the tail vein once a week (each injection was 100 μL).

Cell treatment group (8 animals): Use 0.1 mL of emulsified antigen adjuvant suspension (mixed with a volume of 2 mg / mL chicken type II collagen solution and Freund's complete adjuvant equal volume) for each The root of the mouse tail was injected subcutaneously (the first immunization). After 3 weeks, use 0.1 mL of emulsified antigen adjuvant suspension (mixed with a volume of 2 mg / mL chicken type II collagen solution and Freund's incomplete adjuvant in equal volumes) to each mouse tail root Department underwent subcutaneous injection (second immunization). Starting from the day of the second immunization, the P3 cell preparation prepared in Example 1 was injected into the tail vein once a week (each injection of 2 × 10 ⁶ cells in a volume of 100 μL).

Model group (8 animals): Use 0.1 mL of emulsified antigen adjuvant suspension (mixed with equal volume of chicken type II collagen solution at a concentration of 2 mg / mL and Freund's complete adjuvant in equal volumes). The root of the mouse tail was injected subcutaneously (the first immunization). After 3 weeks, use 0.1 mL of emulsified antigen adjuvant suspension (mixed with a volume of 2 mg / mL chicken type II collagen solution and Freund's incomplete adjuvant in equal volumes) to each mouse tail root Department underwent subcutaneous injection (second immunization). From the day of the second immunization, normal saline was injected into the tail vein once a week (each injection was 100 μL).

Starting from the day of the first immunization, 42 days later, the mice were sacrificed and samples were collected.

2. Phenotype observation

On the 42nd day of the experiment, the paws of each group of mice were photographed to evaluate the swelling of the paws of the mice.

The results are shown in Figure 4 (A is the normal control group, B is the model group, and C is the cell treatment group). Compared with the model group, the paw swelling of the mice in the cell treatment group was significantly reduced, that is, the cell preparation had a significant therapeutic effect on rheumatoid arthritis.

3. Pathological examination of mouse joints

On the 42nd day of the test, the knee joints of the mice were taken to make paraffin sections (section thickness 4 μm), and then hematoxylin-eosin staining was performed.

The results are shown in Figure 5 (A is the normal control group, B is the model group, and C is the cell treatment group). The normal control mice had clear articular cavities, smooth cartilage tissue, and no signs of damage. In the model group, the joint cavity of the mice became narrow, and the synovial hyperplasia was obvious. A large number of inflammatory cells infiltrated, and some cartilage and bone tissue were damaged. In the cell therapy group, the joint cavity of the mice was normal, and the synovial hyperplasia was not obvious. A small amount of inflammatory cells infiltrated, and the cartilage and bone tissue were basically intact.

4. Detection of blood biochemical and immunological indicators

On the 42nd day of the test, blood was collected from the capillary venous plexus behind the eyes of the mice to collect serum. Serum levels of IL-17, TNF-α, IL-6 and IFN-γ were detected. The kit used for the detection of IL-17 is a product of eBioscience, and the article number is 85-88-7170. The kit for detecting IFN-γ is a product of eBioscienc, and the article number is 85-88-7314. The kit for detecting IL-6 is a product of eBioscience, and the article number is 85-88-7064. The kit for detecting TNF-α is a product of eBioscience, and the article number is 85-88-7340.

The detection results of IL-17, TNF-α, IL-6 and IFN-γ are shown in Figure 6. Compared with the normal control group, the levels of inflammatory factors IL-17, TNF-α, IL-6 and IFN-γ in the serum of model mice were significantly increased. Compared with the model group, the levels of IL-17, TNF-α, IL-6 and IFN-γ in the serum of mice in the cell therapy group were significantly reduced.

Industrial applications

The cell preparation provided by the invention is suitable for cell therapy of rheumatoid arthritis and related diseases. The rheumatoid arthritis-related diseases include rheumatoid arthritis complications and diseases with similar pathogenesis, such as rheumatoid vasculitis, pneumonia, osteoarthritis, rheumatoid heart disease, ankylosing spondylitis, Gout, eye disease, kidney disease, urinary system infection, Cushing's syndrome, oral ulcer, etc. The invention has great application value for the treatment of rheumatoid arthritis and related diseases.

Claims (13)

1. Application of mesenchymal stem cells in the preparation of drugs for treating rheumatoid arthritis.
2. The use according to claim 1, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
3. The use according to claim 2, characterized in that the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared by using tissue blocks of the umbilical cord Wharton area as raw materials.
4. The use of cell preparations in the preparation of drugs for the treatment of rheumatoid arthritis;

   The preparation method of the cell preparation includes the following steps: the umbilical cord mesenchymal stem cells are prepared by using the tissue block of the umbilical cord Wharton as a raw material, and a low serum medium is used in the preparation process.
5. The application according to claim 4, wherein the umbilical cord mesenchymal stem cells are primary umbilical cord mesenchymal stem cells or umbilical cord mesenchymal stem cells after passage.
6. The application according to claim 4, wherein the preparation method of the cell preparation comprises the following steps:

   (1) Use low serum medium to culture the isolated umbilical cord Wharton tissue block for 7-10 days;

   (2) After completing step (1), the tissue block is discarded, and the cells are cultured to 80% confluence using a low serum medium;

   (3) After completing step (2), collect the cells, digest them with trypsin, and collect the cells, which are P0 generation umbilical cord mesenchymal cells;

   (4) After step (3) is completed, the P0 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to 80% confluence using a low serum medium;

   (5) After completing step (4), collect the cells, digest them with trypsin, and collect the cells, which are P1 generation umbilical cord mesenchymal cells;

   (6) After completing step (5), the P1 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to a 80% confluence using a low serum medium;

   (7) After completing step (6), collect the cells, digest with trypsin, and collect the cells, which are P2 generation umbilical cord mesenchymal cells;

   (8) After completing step (7), the P2 generation umbilical cord mesenchymal cells are passaged 1: 2, and cultured to 80% confluence using a low serum medium;

   (9) After completing step (8), collect the cells, digest with trypsin, and collect the cells, which is the cell preparation.
7. A medicament for treating rheumatoid arthritis, whose active ingredient is mesenchymal stem cells.
8. The medicine according to claim 7, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
9. The medicament according to claim 8, characterized in that the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared by using tissue blocks of the umbilical cord Wharton area as raw materials.
10. A medicine for treating rheumatoid arthritis, the active ingredient of which is the cell preparation described in claims 4-6.
11. A method for treating rheumatoid arthritis or related diseases, comprising the steps of: administering the drug according to any one of claims 7-10 to a patient with rheumatoid arthritis or a patient with rheumatoid arthritis-related diseases, so that The patient was treated.
12. The method according to claim 11, wherein the rheumatoid arthritis-related diseases include complications of rheumatoid arthritis and diseases with similar pathogenesis.
13. The method according to claim 12, wherein the complications of rheumatoid arthritis and diseases with similar pathogenesis include rheumatoid vasculitis, pneumonia, osteoarthritis, rheumatoid heart disease, ankylosing Spondylitis, gout, eye disease, kidney disease, urinary tract infections, Cushing's syndrome and oral ulcers.

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