A method for inhibiting inflammatory response using fibroblasts

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for inhibiting inflammatory response using fibroblasts. The present invention uses cells separated by digestion of normal foreskin tissue with Clostridium peptidase A as seed cells, and uses serum-free medium for in vitro culture. The obtained fibroblasts have positive expression rates of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, and negative expression rates of CD36, CD39, CD45, and HLA class II molecules less than 5%. The fibroblasts can regulate immune response by secreting TGFβ, inhibit PBMCs proliferation, and thus inhibit inflammatory response, and it has been verified in animal experiments that knee osteoarthritis caused by meniscus injury can be repaired. Therefore, it can be used for the prevention and treatment of inflammatory lesions such as osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for inhibiting inflammatory response by using fibroblasts. Background Art

[0002] Osteoarthritis is a common chronic inflammatory disease that can affect joint tissues throughout the body. The World Health Organization (WHO) predicts that by 2020, osteoarthritis will become the fourth leading cause of disability. Knee osteoarthritis (knee osteoarthritis) refers to a chronic bone and joint disease caused by cartilage degeneration and bone hyperplasia in the knee joint. Clinically, it mainly manifests as knee pain and varying degrees of dysfunction. Some joints have swollen and effusion, which seriously affects the patient's quality of life. X-rays show narrowing of the joint space, osteophyte formation at the joint edge, subchondral bone sclerosis and cystic changes. Knee osteoarthritis is also known as proliferative arthritis of the knee joint, degenerative arthritis and osteoarthritis. It is more common in middle-aged and elderly people, but can also occur in young people. It can be unilateral or bilateral, and more common in women than in men. About 60% of people over 55 years old have knee osteoarthritis on X-rays, of which 35%-50% have clinical manifestations. Generally, articular cartilage begins to degenerate rapidly after the age of 30. With age and frequent use, the cartilage gradually becomes thinner and even ruptures. In addition, obesity is an important risk factor for the development of knee osteoarthritis. Every 5% increase in body weight increases the force on the knee joint by 35%. According to WHO data, there are about 355 million people with knee osteoarthritis worldwide, and it is expected that by 2040, it will affect 25% of the population.

[0003] Knee osteoarthritis can cause joint pain, deformity and dysfunction, which in turn increases the incidence of cardiovascular events and systemic mortality. The existing treatment strategy is to relieve pain, improve joint function and delay disease progression. Currently, the commonly used clinical treatment methods include the following four:

[0004] The first is basic exercise, physical therapy and mobility assistance support, but they can only temporarily relieve the discomfort of the knee joint and cannot improve or stop the progression of knee osteoarthritis. The second is drug treatment, mostly non-steroidal anti-inflammatory drugs (NSAIDs), opioids, corticosteroid injections, etc. It has many adverse reactions and has a certain degree of dependence and drug resistance. The third is the injection of hyaluronic acid, glucocorticoids, etc. into the joint cavity. Although it can relieve pain and improve joint function, it will also aggravate cartilage wear, affect calcium and phosphorus absorption, and reduce pain sensitivity. The 2013 2nd edition of the AAOS Evidence-Based Guidelines for Knee Osteoarthritis strongly recommends against the use of sodium hyaluronate. The fourth is joint replacement: it can improve patient symptoms, but there are many complications such as prosthesis loosening, infection, and periprosthetic fractures.

[0005] In addition to the four commonly used clinical treatment methods, cell therapy is also currently used to treat osteoarthritis, among which mesenchymal stem cells are the main ones. Mesenchymal stem cells (MSCs) have the ability of self-replication, multidirectional differentiation and immune regulation. The MSCs that are currently studied more are mainly derived from bone marrow, but bone marrow-derived MSCs are likely to be highly contaminated by viruses, and their cell number, proliferation, and differentiation capabilities show a significant downward trend with age. The low cell survival rate in the hypoxic and inflammatory environment of the bone joints and cell leakage that may lead to the formation of tumors are the main challenges currently faced by MSCs treatment.

[0006] Liver failure is a serious liver damage caused by multiple factors, which leads to serious damage to the liver's synthesis, metabolism, detoxification and biotransformation functions, causing a large amount of toxic substances to accumulate in the body, causing coagulation dysfunction, hyperbilirubinemia, hepatic encephalopathy and ascites, etc. It is a group of clinical syndromes with the main manifestations. As the pathogenesis of liver failure is not yet very clear, there is a lack of sensitive and standard diagnosis and treatment systems in the clinic, and there are no specific drugs or means for treatment, the treatment of liver failure remains a difficult problem worldwide.

[0007] Traditional comprehensive medical treatment is the basis of liver failure treatment. Although monitoring technology and supportive therapy are constantly improving, the mortality rate of patients is still as high as 60%-80%, among which the mortality rate of acute liver failure is extremely high. Liver transplantation is currently recognized as the only effective method for treating liver failure patients. However, due to the shortage of liver donors, long waiting time, and high cost of transplantation, the number of patients who can actually receive liver transplantation treatment is very limited. In addition, liver failure patients themselves face various difficulties during the perioperative period of liver transplantation. The preoperative liver function is severely damaged, the waiting time for liver donors is long, and there are various complications after transplantation. These are directly related to the short-term efficacy and long-term survival rate of patients. Therefore, seeking new, rapid and effective treatment methods is the focus of liver failure research. How to help liver failure patients safely pass the preoperative waiting period or restore their own liver function without liver transplantation, and effectively deal with complex complications after transplantation is the focus of medical attention. Summary of the invention

[0008] Based on the above requirements, the present invention provides a fibroblast obtained by using cells separated by digestion of normal foreskin tissue with Clostridium peptidase A as seed cells and culturing in vitro with a serum-free medium, wherein the positive expression rates of CD105, CD90, CD73, COL1A1, and FAP are all greater than 80%, and the negative expression rates of CD36, CD39, CD45, and HLA class II molecules are less than 5%. The fibroblast can regulate the immune response by secreting TGFβ, inhibit the proliferation of PBMCs, and thus inhibit the inflammatory response, and it has been verified in animal experiments that the fibroblast can repair knee osteoarthritis caused by meniscus injury, and it has been verified in animal models that the fibroblast can treat chronic acute liver failure induced by CCL4 (carbon tetrachloride) and LPS (lipopolysaccharide).

[0009] The present invention provides a method for preparing fibroblasts, comprising the following steps:

[0010] S1: Primary fibroblasts were obtained by digesting and separating cells from foreskin tissue using Clostridium peptidase A;

[0011] S2: using serum-free medium to culture the primary fibroblasts in vitro;

[0012] S3: The obtained fibroblasts have positive expression rates of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, negative expression rates of CD36, CD39, CD45, and HLA class II molecules less than 5%, and can secrete TGF-β.

[0013] Preferably, the foreskin tissue is discarded foreskin tissue from clinical circumcision surgery, preferably derived from surgical samples of male patients aged no more than 16 years old, and the patients have no infectious viral infection after medical examination.

[0014] More specifically, the method of S1 is:

[0015] S1-1: After washing and disinfecting the foreskin tissue with physiological saline, neutral proteinase A was added, and after incubation, the tissue was washed with precooled physiological saline, the dermis and epidermis of the tissue were mechanically separated, the epidermis was removed, the dermis was cut into pieces as much as possible, washed with precooled physiological saline, and Clostridium peptidase A was added to incubate to obtain primary fibroblasts;

[0016] More specifically, the foreskin tissue was cleaned and disinfected with physiological saline, and then neutral proteinase A with a final concentration of 2DMC U / mL was added, and the tissue was incubated at 37°C for 16-20 hours, and then the tissue was washed with precooled physiological saline for 1-2 times. The dermis and epidermis of the tissue were separated with sterilized tweezers, and the epidermis was removed. The dermis was cut into pieces as small as 0.5-2 mm, and then the tissue was washed with precooled physiological saline for 1-2 times, and then the tissue was transferred to a 50 ml centrifuge tube, and a final concentration of 0.1PZ U / mL Clostridium peptidase A was added, and the tissue was incubated at 37°C for 2-3 hours.

[0017] S1-2: Use a 70-micron cell filter to screen the cell suspension obtained in S1-11 with the aid of physiological saline, collect the cell suspension under the cell filter, centrifuge and discard the supernatant, wash by centrifugation with serum-free medium, and resuspend with serum-free medium to obtain a primary cell suspension;

[0018] Further specific operations are: using a 70-micron cell filter to screen the cell suspension with the aid of physiological saline, collecting the cell suspension under the cell filter, centrifuging at 1000 rpm for 5 minutes, discarding the supernatant, washing 1-2 times by centrifugation with serum-free medium, and resuspending with serum-free medium to obtain a primary cell suspension;

[0019] S1-3: Use a 40-micron cell filter to perform sieve sorting on the primary cell suspension with the aid of physiological saline, collect the cell suspension harvested by filtration under the cell filter to complete the sieve sorting, centrifuge the obtained filtrate and remove the supernatant to obtain primary fibroblasts.

[0020] In addition, in a specific implementation manner, the method of S2 is:

[0021] The primary fibroblasts are expanded and cultured in a serum-free medium until the cell confluence is not less than 80%, and then digested with a trypsin digestion solution, and then subcultured with a serum-free medium to obtain fibroblasts;

[0022] Preferably, the serum-free culture medium is high-glucose DMEM, with hydrocortisone, bFGF and human platelet lysate added.

[0023] More specifically, the method of S2: the trypsin digestion solution is digested for 1-5 minutes; the concentration of hydrocortisone is 5-25ng / mL, the concentration of bFGF is 5-30ng / mL, and the human platelet lysate is 4-10%; preferably, the concentration of hydrocortisone is 10-20ng / mL, the concentration of bFGF is 15-25ng / mL, and the human platelet lysate is 6-9%.

[0024] The present invention further provides fibroblasts obtained by the method, wherein the fibroblasts positively express CD105, CD90, CD73, COL1A1, and FAP at an expression rate of more than 80%, negatively express CD36, CD39, CD45, and HLA class II molecules at an expression rate of less than 5%, and can secrete TGF-β.

[0025] The present invention further provides the use of the fibroblasts in the preparation of a drug for treating an inflammatory disease. Preferably, the inflammatory disease is local inflammation of osteoarthritis or acute liver failure.

[0026] Particularly, the diseases are osteoarthritis, acute liver failure.

[0027] The present invention thus also provides a pharmaceutical composition containing the fibroblasts as an active ingredient. Specifically, it is a dosage form suitable for injection, such as intra-articular injection, intramuscular injection, and intravenous injection. When used for the treatment of osteoarthritis, the cell suspension can be directly injected into the joint cavity with a microinjection needle, and the injection volume is less than 1 mL.

[0028] The present invention uses cells separated by digestion of normal foreskin tissue as seed cells, and uses serum-free culture medium for in vitro culture, wherein hydrocortisone, bFGF and human platelet lysate are added to the culture medium. bFGF (fibroblast growth factor) regulates the proliferation of fibroblasts, can significantly increase the proliferation rate of fibroblasts, and improve the expression of fibroblast collagen, hydrocortisone can significantly shorten the doubling time of fibroblasts, and human platelet lysate can promote cell adhesion and proliferation as a serum substitute. The fibroblasts thus cultured have positive expression rates of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, and negative expression rates of CD36, CD39, CD45, and HLA class II molecules less than 5%. The fibroblasts can regulate immune responses by secreting TGFβ, inhibit PBMCs proliferation, and thus inhibit inflammatory responses. Animal experiments have proven that they can repair knee osteoarthritis caused by meniscus injury, and animal models have proven that they can treat chronic acute liver failure induced by CCL4 (carbon tetrachloride) and LPS (lipopolysaccharide). Therefore, they can be used for the prevention and treatment of diseases such as osteoarthritis and liver failure.

[0029] The human fibroblast injection for the treatment of osteoarthritis and liver failure meets the urgent needs of the market. The present invention has the following advantages: ① Avoiding ethical restrictions: Compared with mesenchymal stem cells derived from embryonic stem or bone marrow stem, foreskin is medical waste, without ethical restrictions, widely available, easy to obtain, and harmless to the donor; ② Helping patients with liver failure to survive the acute phase; ③ The transplantation method is simple and relieves the pain of patients: The cells are directly injected into the joint cavity or intravenously, the operation is easy, the wound caused to the patient is small, and the pain of the patient is relieved; ④ High safety and not easy to cause infection: Fibroblasts are non-immunogenic, non-pathogen microbial infection, and are not prone to adverse reactions such as postoperative infection in patients; ⑤ No matching and no immune rejection: Fibroblasts are non-immunogenic, injection does not require matching, and patients do not need to use immunosuppressants; ⑥ Stable quality and clear effect: Fibroblast injection is managed and prepared according to drug management, with large output and small differences in effects between batches, and can be used by patients multiple times at any time. The fibroblasts provided by the present invention can be allogeneically infused and amplified on a large scale in vitro. Therefore, the present invention has great application prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Flow chart for the isolation and culture of fibroblasts.

[0031] Figure 2 This is a diagram of the fibroblast morphology.

[0032] FIG. 3A to FIG. 3I They are result graphs showing that the positive cell markers CD90, FAP, COL1A1, CD105, and CD73 positivity rates are greater than 80% in flow cytometry, and the negative cell markers CD36, CD39, CD45, and HLA-DRPQ positivity rates are less than 5%.

[0033] Figure 4A This is the inhibition of PBMCs proliferation by fibroblasts after co-culture of donor 23019 cells with activated PBMCs.

[0034] Figure 4B This is the inhibition of PBMCs proliferation by fibroblasts after co-culture of donor 23021 cells with activated PBMCs.

[0035] Figure 4C This is the inhibition of PBMC proliferation by fibroblasts after co-culture of MSC cells with activated PBMCs.

[0036] Figure 4D The PBMCs unactivated control was used as the PBMCs cell status of the negative group.

[0037] Figure 4E The cell status of PBMCs was used as a positive control for PBMCs activation.

[0038] Figure 5 This is the secretion of TGFβ by fibroblasts.

[0039] Figure 6 The secretion of CXCL12 by fibroblasts.

[0040] Fig. 7A The left knee joint tissue was stained in the normal group.

[0041] Figure 7B Left knee joint tissue staining of the experimental group.

[0042] Figure 7C Left knee joint tissue staining of the model group.

[0043] Fig.7D This is the staining of the right knee joint tissue of the normal group.

[0044] Fig. 7E Right knee joint tissue staining in the experimental group.

[0045] Figure 7F The right knee joint tissue of the model group was stained.

[0046] Fig. 8A This is the result of alanine aminotransferase test.

[0047] Figure 8B This is the result of aspartate aminotransferase test.

[0048] Figure 8C The survival curves of the three groups of animals are shown in Figure 2.

[0049] Fig.8D The livers of the normal group animals were sampled and then stained pathologically.

[0050] Fig. 8E The livers of the model group animals were sampled and then subjected to pathological staining.

[0051] Figure 8F The livers of the experimental group animals were sampled and then subjected to pathological staining. DETAILED DESCRIPTION

[0052] The present invention is further described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0053] Example 1: Using serum-free medium to culture seed cells in vitro to obtain primary cells 1. Statement of the nature of the starting tissue and legality of the source:

[0054] like Figure 1As shown in the flow chart of fibroblast isolation and culture, the present invention uses cells separated from normal foreskin tissue by digestion with Clostridium peptidase A as seed cells, and uses serum-free culture medium for in vitro culture. The obtained fibroblasts positively express CD105, CD90, CD73, COL1A1, and FAP, and negatively express CD36, CD39, CD45, and HLA class II molecules.

[0055] Among them, in this embodiment, the foreskin tissue removed during the circumcision is normal skin tissue, and is a surgical sample from a male patient aged no older than 16 years old. The patient has no infectious viral infection after medical examination. The patient is fully informed of the purpose of obtaining the surgical sample before the operation, and the patient himself or the guardian has signed an informed consent form.

[0056] 2. Obtaining primary cells:

[0057] First, use physiological saline to clean and disinfect the foreskin tissue, add neutral proteinase A with a final concentration of 2DMC U / mL, incubate at 37°C for 16-20 hours, wash 1-2 times with pre-cooled physiological saline, use sterilized tweezers to separate the dermis and epidermis of the tissue, remove the epidermis, cut the dermis into pieces as small as 0.5-2mm, wash 1-2 times with pre-cooled physiological saline, transfer to a 50ml centrifuge tube, add Clostridium peptidase A with a final concentration of 0.1PZ U / mL, and incubate at 37°C for 2-3 hours.

[0058] Then, the cell suspension was screened with a 70-micron cell filter with the aid of physiological saline, and the filtrate was collected and the mucus and undigested tissue that were not filtered out of the cell filter were removed. Then, the cell suspension under the cell filter was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell suspension was washed 1-2 times by centrifugation with serum-free medium, and the primary cell suspension was obtained after resuspending with serum-free medium.

[0059] Then, the primary cell suspension was sieved using a 40-micron cell filter with the aid of physiological saline, and the cell suspension harvested by filtration under the cell filter was collected to complete the sieve sorting. The obtained filtrate was centrifuged and the supernatant was removed to obtain the primary cells.

[0060] Example 2: A serum-free culture medium was used to culture a cell suspension of primary fibroblasts in vitro to obtain target fibroblasts.

[0061] The specific steps of in vitro culture are:

[0062] Primary fibroblasts were placed in a 6-well cell culture plate at a seeding area of ​​15,000 cells / cm2. 2 ml of serum-free medium was added to each well to expand and culture until the cell confluence was not less than 80%. 1 ml of recombinant trypsin (TrypLE TM ) digestion solution for 1-5 minutes, and then continue to use serum-free medium for subculture. The serum-free medium is high-glucose DMEM, with 15ng / mL hydrocortisone, 20ng / mL bFGF, and 7.5% human platelet lysate added.

[0063] The primary cells were placed in a 6-well plate at an inoculation area of ​​15,000 cells / cm2, and 2 ml of serum-free medium was added to each well to expand and culture until the cell confluence was not less than 80%. After digestion with recombinant trypsin digestion solution for 1-5 minutes, serum-free medium was continued for subculture, and subculture was continued. Among them, the fibroblasts obtained in this example have a morphology as shown in FIG. Figure 2 shown.

[0064] Example 3: Fibroblast-related detection and analysis

[0065] 1. Perform surface antibody flow cytometry detection on the obtained fibroblasts.

[0066] After discarding the obtained fibroblast culture medium, rinse with sterile PBS buffer, then digest with recombinant trypsin digestion solution and collect the precipitate by centrifugation; add 100 μl of staining buffer to the precipitate to resuspend the cells in the flow tube, then add the flow cytometry antibodies to be tested and incubate for 30 minutes, then resuspend each tube with 400 μl of staining buffer and perform surface antibody flow cytometry detection.

[0067] The flow cytometry results are as follows FIG. 3A to FIG. 3I The results showed that the positive cell markers with a positive rate greater than 70% included: CD90, FAP, COL1A1, CD105, and CD73.

[0068] 2. In the in vitro fibroblast experiment, it can inhibit the proliferation of PBMCs.

[0069] The activated PBMCs cells were co-cultured with fibroblasts in a 24-well culture plate. After 96 hours of culture, the proliferation of the PBMCs cells was observed. The activated PBMCs cells proliferated in the form of cell spheres. The fibroblasts could inhibit the proliferation of the activated PBMCs, preventing the PBMCs cells from proliferating in the form of cell spheres. Since MSCs (mesenchymal stem cells) are believed to have the ability to inhibit the proliferation of PBMCs, they were used as the positive control group.

[0070] The results are as follows FIG. 4A to FIG. 4E shown. Figure 4EFor the positive control (PBMCs activation control) group, PBMCs proliferated in sphere-like manner; Figure 4D The negative (PBMCs unactivated control) group, PBMCs cells did not proliferate; Figure 4A The 23019-PBMCs group was a co-culture of donor 23019 cells and activated PBMCs cells, and the PBMCs cells were in a non-proliferative state like the non-activated PBMCs group; Figure 4B The 23021-PBMCs group was a co-culture of donor 23021 cells and activated PBMCs cells, and the PBMCs cells were in a non-proliferative state like the non-activated PBMCs group; Figure 4C The MSC-PBMCs group consisted of MSC cells co-cultured with activated PBMCs cells, and the PBMCs cells were in a non-proliferative state like the non-activated PBMCs group.

[0071] 3. Secretion of transforming growth factor (TGFβ) by fibroblasts

[0072] The culture supernatants of fibroblasts from three donors and different pronouns were collected, and the TGFβ content in the supernatant was detected using a TGFβ detection kit, and converted into the TGFβ content that can be secreted per 1 million cells in 24 hours.

[0073] The results are as follows Figure 5 As shown, it can be seen that fibroblasts from different donor sources can maintain a high level of TGFβ secretion, and will not change significantly with cell passage. TGF-β can inhibit the proliferation and differentiation of Th1, Th2, and CTL, thereby inhibiting local inflammatory response. It can inhibit the proliferation of PBMCs in cell experiments and treat knee osteoarthritis in animal experiments.

[0074] 4. Secretion of human stromal cell-derived factor (CXCL12) by fibroblasts

[0075] The culture supernatants of fibroblasts from three donors and different pronouns were collected, and the CXCL12 content in the supernatant was detected using a CXCL12 detection kit, and converted into the CXCL12 content that can be secreted per 1 million cells in 24 hours.

[0076] The results are as follows Figure 6As shown, it can be seen that fibroblasts from different donor sources can maintain a high level of CXCL12 secretion, and will not change significantly with cell passage. Although low concentrations of CXCL12 can recruit immune cells, high concentrations of CXCL12 can repel CD4+ and CD8+ T cells, inhibit T cells from enriching locally, and reduce local immune responses. In cell experiments, it can inhibit the proliferation of PBMCs, and in animal experiments, it can treat knee osteoarthritis.

[0077] Example 4: Application of fibroblasts

[0078] 1. Osteoarthritis modeling method

[0079] Select male SD rats of 9-10 weeks of age, body weight 250-300g as research object, after rat is anesthetized with isoflurane, knee joint is shaved, and supine position is fixed on operating board and continues anesthesia. Knee joint disinfection, cut right knee joint position skin and muscle successively, free knee joint, and cross section the anterior cruciate ligament of knee joint. Then sew muscle and skin, after wiping disinfection with iodine tincture, be positioned over heating pad rewarming, put back cage box and raise after it wakes up. The cross ligament cross section group of the knee joint of rat is model group, and the knee joint that is not processed is normal group, and the fibroblast group that cross ligament cross section and injection into embodiment two obtain is experimental group.

[0080] 2. Cell injection

[0081] Two weeks after surgery, the test substance (fibroblasts) was taken out of the liquid nitrogen tank and transported on dry ice; the test substance was immediately immersed in a 37°C water bath and gently shaken until the liquid was completely melted, and then shaken left and right. All the liquid was taken out with a pipette and added to a 50mL sterile centrifuge tube, and the centrifuge was set to 200g for 5 minutes. After centrifugation, the supernatant was discarded and the cells were diluted to 2E+07 / mL with saline. Using a microinjection needle, 1E+06 cells (50 microliters) were injected into the joint cavity. The knee joint injected with the test substance was the experimental group.

[0082] 3. Data Collection

[0083] Rats were treated with CO 28 days after the first treatment. 2 After euthanasia, the right and left knee joints were dissected and sent to a third party for HE staining.

[0084] 4. Experimental results

[0085] like Figures 7A to 7F As shown, Fig. 7A and 7D : The left and right knee joint tissues of the normal group are stained. The joints are relatively smooth and without damage. Figure 7B and 7E:The left and right knee joint tissues of the experimental group were stained. There was damage in the joints, but Figure 7C and 7F Compared with the modeling group, the experimental group injected with fibroblasts had better repair of joint damage. Fibroblasts can better repair knee vallecular arthritis caused by ligament transection.

[0086] 5. Acute-on-chronic liver failure modeling method

[0087] Male SD rats aged 9-10 weeks and weighing 250-300 g were selected as research subjects. 10% CCL4 was injected into the rat peritoneum at 2 mL / kg twice a week for 12 consecutive weeks. At the 13th week, LPS was injected into the peritoneum at 1 mg / kg at the same time. The modeling group did not receive treatment, and the experimental group was injected with the fibroblasts obtained in Example 2 after LPS injection.

[0088] 6. Cell Injection

[0089] The test substance (fibroblasts) was taken out of the liquid nitrogen tank and placed on dry ice for transportation; the test substance was immediately immersed in a 37°C water bath, gently shaken until the liquid was completely melted, and shaken left and right. All the liquid was taken out with a pipette and added to a 50mL sterile centrifuge tube, and the centrifuge was set to 200g for 5 minutes. After the centrifugation, the supernatant was discarded, and the cells were diluted to 2E+07 / mL with saline. After the rat was anesthetized with isoflurane, it was fixed on the operating board in the supine position and anesthetized continuously. The portal vein was freed and 1E+07 cells (1000 microliters) were injected into the portal vein with an injection needle.

[0090] 7. Data Collection

[0091] Rats were treated with CO 3 days after the first treatment. 2 After euthanasia, liver tissue was obtained through autopsy and sent to a third party for HE staining, and blood was taken for transaminase testing.

[0092] 8. Experimental results

[0093] like Figures 8A-8F As shown, the normal group was male rats, the model group was male rats modeled with CCL4 and LPS, and the experimental group was a group injected with fibroblasts after modeling with CCL4 and LPS. Fig. 8A The results of alanine aminotransferase test after blood collection showed that the alanine aminotransferase in the modeling group increased significantly, while that in the experimental group decreased significantly, approaching the value in the normal group. Figure 8B The results of aspartate aminotransferase test after blood sampling showed that the aspartate aminotransferase in the modeling group increased significantly, while the aspartate aminotransferase in the experimental group decreased significantly, approaching the value of the normal group; Figure 8CThe figure is the survival curve of three groups of animals. The initial number of each group is 10. No animals died in the normal group, 5 animals died in the model group, and 2 animals died in the experimental group. Fig.8D The results of pathological staining after liver sampling in the normal group of animals; Fig. 8E The results of pathological staining after liver sampling in the model group animals; Figure 8F These are the results of pathological staining after liver sampling from the experimental group animals.

Claims

1. Use of fibroblasts in the preparation of a drug for treating osteoarthritis or acute-on-chronic liver failure, characterized in that: The method for preparing fibroblasts comprises the following steps: S1: Primary fibroblasts were obtained by digesting and separating cells from foreskin tissue using Clostridium peptidase A; S2: After the primary fibroblasts are expanded and cultured in a serum-free medium until the cell confluence is not less than 80%, they are digested with trypsin digestion solution, and then subcultured with a serum-free medium to obtain fibroblasts; the serum-free medium is a high-glucose DMEM with hydrocortisone, bFGF and human platelet lysate added thereto; The concentration of hydrocortisone is 5-25 ng / mL, the concentration of bFGF is 5-30 ng / mL, and the concentration of human platelet lysate is 4-10%; S3: The obtained fibroblasts have positive expression rates of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, negative expression rates of CD36, CD39, CD45, and HLA class II molecules less than 5%, and can secrete TGF-β.

2. The use according to claim 1, characterized in that The foreskin tissue is the foreskin tissue discarded from clinical circumcision surgery.

3. The use according to claim 2, characterized in that The foreskin tissue is a surgical sample from a male patient no older than 16 years old, and the patient has been found to have no infectious viral infection through medical examination.

4. The use according to claim 1, characterized in that The method of S1 is: S1-1: After washing and disinfecting the foreskin tissue with physiological saline, neutral proteinase A was added, and after incubation, the tissue was washed with precooled physiological saline, the dermis and epidermis of the tissue were mechanically separated, the epidermis was removed, the dermis was cut into pieces as much as possible, washed with precooled physiological saline, and Clostridium peptidase A was added to incubate to obtain primary fibroblasts; S1-2: Use a 70-micron cell filter to screen the cell suspension obtained in S1-1 with the aid of physiological saline, collect the cell suspension under the cell filter, centrifuge and discard the supernatant, wash by centrifugation with serum-free medium, and resuspend with serum-free medium to obtain a primary cell suspension; S1-3: Use a 40-micron cell filter to perform sieve sorting on the primary cell suspension with the aid of physiological saline, collect the cell suspension harvested by filtration under the cell filter to complete the sieve sorting, centrifuge the obtained filtrate and remove the supernatant to obtain primary fibroblasts.

5. The use according to claim 4, characterized in that The method of S1 is: S1-1: After cleaning and disinfecting the foreskin tissue with saline, add neutral proteinase A with a final concentration of 2DMC U / mL, incubate at 37°C for 16-20 hours, wash 1-2 times with pre-cooled saline, use sterilized tweezers to separate the dermis and epidermis of the tissue, remove the epidermis, cut the dermis as small as possible to 0.5-2mm, wash 1-2 times with pre-cooled saline, transfer to a 50ml centrifuge tube, add Clostridium peptidase A with a final concentration of 0.1PZ U / mL, and incubate at 37°C for 2-3 hours; S1-2: Use a 70-micron cell filter to sieve the cell suspension obtained in S1-1 with the aid of physiological saline, collect the cell suspension under the cell filter, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash 1-2 times by centrifugation with serum-free medium, and resuspend in serum-free medium to obtain the primary cell suspension.

6. The use according to claim 1, characterized in that In S2: the trypsin digestion solution is digested for 1-5 minutes; the concentration of hydrocortisone is 10-20 ng / mL, the concentration of bFGF is 15-25 ng / mL, and the concentration of human platelet lysate is 6-9%.

7. The use according to any one of claims 1 to 6, characterized in that The drug is in a dosage form suitable for injection.

8. The use according to claim 7, characterized in that The medicine is in a dosage form suitable for intra-articular injection, intramuscular injection or intravenous injection.

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