Method for inhibiting inflammatory response by using fibroblast

Abstract

The present invention relates to the technical field of biology, and particularly relates to a method for inhibiting an inflammatory response by using a fibroblast. In the present invention, a fibroblast, which is obtained by digesting normal foreskin tissue by means of clostridiopeptidase A to isolate a cell as a seed cell and culturing the seed cell in vitro by using a serum-free medium, shows positive expression of CD105, CD90, CD73, COL1A1, and FAP, with expression rates all exceeding 80%, and negative expression of CD36, CD39, CD45, and HLA class II molecules, with expression rates below 5%. The fibroblast can regulate an immune response by secreting TGFβ, inhibiting proliferation of PBMCs, thereby inhibiting the inflammatory response, and has been verified in an animal experiment to have the ability to repair knee osteoarthritis caused by meniscus injury. Therefore, the fibroblast can be used for preventing and treating inflammatory lesions such as osteoarthritis.

Description

A method for inhibiting inflammatory response using fibroblasts Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a method for inhibiting inflammatory response using fibroblasts. Background Art

[0002] Osteoarthritis is a common chronic inflammatory disease that can affect joints throughout the body. The World Health Organization (WHO) predicts that by 2020, osteoarthritis will become the fourth leading cause of disability. Knee osteoarthritis (KOA) is a chronic bone and joint disorder caused by cartilage degeneration and bone hyperplasia in the knee joint. Clinically, it presents with knee pain and varying degrees of functional impairment, sometimes with joint swelling and effusion, severely impacting patients' quality of life. X-ray findings include joint space narrowing, osteophyte formation at the joint margins, subchondral bone sclerosis, and cystic changes. Knee OA, also known as proliferative arthritis of the knee, degenerative arthritis, and osteoarthritis, is more common in middle-aged and elderly individuals, but can also occur in younger individuals. It can be unilateral or bilateral, and is more common in women than in men. Approximately 60% of people over 55 years old show evidence of knee OA on X-ray, and 35%-50% of these individuals experience clinical symptoms. Generally, articular cartilage begins to degenerate rapidly after the age of 30. With aging and frequent use, the cartilage gradually thins and may even rupture and become defective. Furthermore, obesity is a significant risk factor for the development of knee osteoarthritis. Every 5% increase in body weight increases the forces exerted on the knee joint by 35%. According to the World Health Organization, approximately 355 million people worldwide suffer from knee osteoarthritis, and it is projected to affect 25% of the population by 2040.

[0003] Knee osteoarthritis can cause joint pain, deformity, and mobility impairment, which in turn increases the incidence of cardiovascular events and overall mortality. Existing treatment strategies are aimed at relieving pain, improving joint function, and delaying disease progression. Currently, the following four treatment methods are commonly used in clinical practice:

[0004] The first is basic exercise, physical therapy and mobility assistance support, but these 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 surgery: it can improve patient symptoms, but there are many complications such as prosthesis loosening, infection, and peri-prosthetic 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. However, due to the possibility of high viral contamination of bone marrow-derived MSCs, and the obvious downward trend in their cell number, proliferation and differentiation ability with age, the low cell survival rate in the hypoxic and inflammatory environment of the bones and 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 severe liver damage caused by multiple factors, resulting in severe impairment of the liver's synthesis, metabolism, detoxification, and biotransformation functions. This leads to the accumulation of toxic substances in the body, resulting in a clinical syndrome characterized by coagulation disorders, hyperbilirubinemia, hepatic encephalopathy, and ascites. Due to the lack of a clear pathogenesis, a sensitive and standardized clinical diagnosis and treatment system, and the absence of effective medications or treatments, the treatment of liver failure remains a daunting challenge worldwide.

[0007] Traditional comprehensive medical treatment is the cornerstone of liver failure treatment. Despite continuous improvements in monitoring techniques and supportive therapies, the mortality rate remains as high as 60%-80%, with acute liver failure being particularly fatal. Liver transplantation is currently recognized as the only effective treatment for liver failure patients. However, due to a shortage of donor livers, long waiting times, and high transplant costs, the number of patients who can actually receive a liver transplant is very limited. Furthermore, liver failure patients face numerous challenges during the perioperative period, including severe preoperative liver impairment, long waiting times for donor livers, and the potential for various post-transplant complications. These challenges directly impact both the short-term efficacy of the transplant and long-term survival. Therefore, the search for new, rapid, and effective treatments is a key focus of liver failure research. Helping patients with liver failure safely navigate the preoperative waiting period, recovering their liver function to eliminate the need for a liver transplant, and effectively managing the complex complications after transplantation remain key medical concerns. Summary of the invention:

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

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

[0010] S1: Primary fibroblasts were obtained by digesting and isolating foreskin tissue with Clostridium peptidase A.

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

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

[0013] Preferably, the foreskin tissue is discarded foreskin tissue from a clinical circumcision operation, preferably derived from a surgical sample of a male patient no older than 16 years old, and the patient has been medically examined to be free of infectious viral infection.

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

[0015] S1-1: After washing and disinfecting the foreskin tissue with normal saline, neutral proteinase A was added, incubated, and then washed with pre-cooled normal saline. The dermis and epidermis of the tissue were mechanically separated, the epidermis was removed, and the dermis was minced as much as possible, washed with pre-cooled normal saline, and incubated with Clostridium peptidase A to obtain primary fibroblasts;

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

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

[0018] The specific operation is as follows: the cell suspension is screened using a 70-micron cell strainer with the aid of physiological saline, the cell suspension under the cell strainer is collected, centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and the cell suspension is washed 1-2 times by centrifugation with serum-free medium, and resuspended in serum-free medium to obtain a primary cell suspension;

[0019] S1-3: Using a 40-μm cell strainer, the primary cell suspension is subjected to sieve sorting with the aid of physiological saline, and the cell suspension harvested by filtration under the cell strainer is collected to complete sieve sorting. The obtained filtrate is centrifuged and the supernatant is removed to obtain primary fibroblasts.

[0020] In addition, in a specific embodiment, 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 trypsin digestion solution, and then continued to be subcultured in 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 S2 method: the trypsin digestion solution is digested for 1-5 minutes; the concentration of hydrocortisone is 5-25 ng / mL, the concentration of bFGF is 5-30 ng / mL, and the human platelet lysate is 4-10%; preferably, the concentration of hydrocortisone is 10-20 ng / mL, the concentration of bFGF is 15-25 ng / mL, and the human platelet lysate is 6-9%.

[0024] The present invention further provides fibroblasts obtained by the method, wherein the fibroblasts have a positive expression rate of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, a negative expression rate of CD36, CD39, CD45, and HLA class II molecules 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 also provides a pharmaceutical composition containing the fibroblasts as an active ingredient. Specifically, the composition is in a dosage form suitable for injection, such as intra-articular injection, intramuscular injection, or intravenous injection. For the treatment of osteoarthritis, the cell suspension can be directly injected into the joint cavity using a microinjection needle, with an injection volume of less than 1 mL.

[0028] The present invention uses cells isolated from digested normal foreskin tissue as seed cells and cultured in vitro in a serum-free medium supplemented with hydrocortisone, bFGF, and human platelet lysate. bFGF (fibroblast growth factor) regulates fibroblast proliferation, significantly increasing its proliferation rate and enhancing its collagen expression. Hydrocortisone significantly shortens the doubling time of fibroblasts. Human platelet lysate, acting as a serum substitute, promotes cell adhesion and proliferation. The fibroblasts obtained by this culture showed 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 below 5%. These fibroblasts can regulate immune responses by secreting TGFβ, inhibiting the proliferation of PBMCs, and thus suppressing inflammation. Animal experiments have shown that they can repair knee osteoarthritis caused by meniscus injury and treat chronic acute liver failure induced by CCL4 (carbon tetrachloride) and LPS (lipopolysaccharide) in animal models. Therefore, they can be used for the prevention and treatment of diseases such as osteoarthritis and liver failure.

[0029] Human fibroblast injection for the treatment of osteoarthritis and liver failure meets urgent market needs. The present invention has the following advantages: 1. Avoids ethical restrictions: Compared with mesenchymal stem cells derived from embryonic stem cells or bone marrow stem cells, foreskin is medical waste, free of ethical restrictions, widely available, and harmless to the donor; 2. Helps patients with liver failure survive the acute phase; 3. Simple transplantation method, alleviating patient pain: Direct injection of cells into the joint cavity or intravenously, with low surgical difficulty and minimal incision, alleviating patient pain; 4. High safety, low risk of infection: Fibroblasts are non-immunogenic and non-infectious to pathogenic microorganisms, making them less susceptible to adverse reactions such as postoperative infection; 5. No need for matching, no immune rejection: Fibroblasts are non-immunogenic, and no matching is required for injection, eliminating the need for immunosuppressants; 6. Stable quality, clear efficacy: Fibroblast injection is managed and prepared according to drug regulations, resulting in high yield, minimal variability in efficacy between batches, and ready for multiple use by patients. The fibroblasts provided by the present invention can be allogeneically infused and expanded on a large scale in vitro. Therefore, the present invention has great application prospects and application value. Description of the drawings:

[0030] Figure 1 is a flow chart of fibroblast isolation and culture.

[0031] Figure 2 shows the morphology of fibroblasts.

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

[0033] FIG4A shows the inhibition of PBMC proliferation by fibroblasts after co-culture of donor 23019 cells with activated PBMCs.

[0034] FIG4B shows the inhibition of PBMC proliferation by fibroblasts after co-culture of donor 23021 cells with activated PBMCs.

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

[0036] FIG4D shows the cell status of PBMCs in the negative group as a non-activated PBMCs control.

[0037] FIG4E shows the cell status of PBMCs activated as a positive control.

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

[0039] FIG6 shows the secretion of CXCL12 by fibroblasts.

[0040] Figure 7A shows the staining of the left knee joint tissue in the normal group.

[0041] Figure 7B shows the staining of the left knee joint tissue in the experimental group.

[0042] Figure 7C shows the staining of the left knee joint tissue in the model group.

[0043] Figure 7D shows the staining of the right knee joint tissue in the normal group.

[0044] Figure 7E shows the staining of the right knee joint tissue in the experimental group.

[0045] Figure 7F shows the staining of the right knee joint tissue in the model group.

[0046] FIG8A shows the results of alanine aminotransferase detection.

[0047] FIG8B shows the results of aspartate aminotransferase detection.

[0048] FIG8C is a survival curve of the three groups of animals.

[0049] Figure 8D shows the pathological staining of liver samples from animals in the normal group.

[0050] Figure 8E shows the pathological staining of liver samples from animals in the model group.

[0051] Figure 8F shows the pathological staining of liver samples from the experimental group animals. Specific implementation method:

[0052] The present invention will be further described below through specific embodiments 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

[0054] 1. Statement on the nature of the originating organization and the legitimacy of the source:

[0055] As shown in the flow chart of fibroblast isolation and culture in Figure 1, the present invention uses cells separated from normal foreskin tissue by digestion with Clostridium peptidase A as seed cells and cultured in vitro using serum-free medium. The obtained fibroblasts positively express CD105, CD90, CD73, COL1A1, and FAP, and negatively express CD36, CD39, CD45, and HLA class II molecules.

[0056] 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 more 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 his guardian has signed an informed consent form.

[0057] 2. Acquisition of primary cells:

[0058] First, clean and disinfect the foreskin tissue with normal 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 normal 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 normal 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.

[0059] The cell suspension was then screened using a 70-μm cell strainer with the aid of physiological saline. The filtrate was collected and any mucus and undigested tissue that remained above the strainer was removed. The cell suspension below the strainer was then centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells were washed 1-2 times by centrifugation with serum-free medium. The cells were then resuspended in serum-free medium to obtain a primary cell suspension.

[0060] Then, the primary cell suspension was screen-sorted using a 40-μm cell strainer with the aid of physiological saline, and the cell suspension harvested by filtration under the cell strainer was collected to complete the screen sorting. The obtained filtrate was centrifuged and the supernatant was removed to obtain the primary cells.

[0061] Example 2: A cell suspension of primary fibroblasts was cultured in vitro using a serum-free culture medium to obtain target fibroblasts.

[0062] The specific steps of in vitro culture are:

[0063] Primary fibroblasts were placed in a 6-well cell culture plate at an inoculation 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 subculture in serum-free medium. The serum-free medium is high-glucose DMEM supplemented with 15 ng / mL hydrocortisone, 20 ng / mL bFGF, and 7.5% human platelet lysate.

[0064] Primary cells were plated at a seeding density of 15,000 cells / cm² in a 6-well plate. 2 ml of serum-free medium was added to each well for expansion and culture until the cell confluence reached no less than 80%. The cells were then digested with recombinant trypsin for 1-5 minutes and then subcultured in serum-free medium. The morphology of the fibroblasts obtained in this example is shown in FIG2 .

[0065] Example 3: Fibroblast-related detection and analysis

[0066] 1. Perform surface antibody flow cytometry detection on the obtained fibroblasts.

[0067] The obtained fibroblast culture medium was discarded and rinsed with sterile PBS buffer, then digested with recombinant trypsin digestion solution and centrifuged to collect the precipitate; 100 μl of staining buffer was added to the precipitate to resuspend the cells in a flow cytometry tube, and the flow cytometry antibodies to be tested were added and incubated for 30 minutes. After that, each tube was resuspended with 400 μl of staining buffer and subjected to surface antibody flow cytometry detection.

[0068] The flow cytometry results are shown in Figures 3A to 3I. The results showed that the positive cell markers with a positive rate greater than 70% included: CD90, FAP, COL1A1, CD105, and CD73.

[0069] 2. In the in vitro fibroblast experiment, it can inhibit the proliferation of PBMCs.

[0070] Activated PBMCs were mixed with fibroblasts and co-cultured in a 24-well culture plate. After 96 hours of culture, the proliferation of PBMCs was observed. Activated PBMCs proliferated in the form of cell spheres. Fibroblasts inhibited the proliferation of activated PBMCs, preventing them 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 a positive control group.

[0071] The results are shown in Figures 4A to 4E. Figure 4E shows the positive control (PBMCs activated control) group, in which PBMCs proliferated into spheres; Figure 4D shows the negative (PBMCs unactivated control) group, in which PBMCs did not proliferate; Figure 4A shows the 23019-PBMCs group, in which donor 23019 cells were co-cultured with activated PBMCs, and the PBMCs showed the same non-proliferation state as the unactivated PBMCs group; Figure 4B shows the 23021-PBMCs group, in which donor 23021 cells were co-cultured with activated PBMCs, and the PBMCs showed the same non-proliferation state as the unactivated PBMCs group; and Figure 4C shows the MSC-PBMCs group, in which MSCs were co-cultured with activated PBMCs, and the PBMCs showed the same non-proliferation state as the unactivated PBMCs group.

[0072] 3. Secretion of transforming growth factor (TGFβ) by fibroblasts

[0073] 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.

[0074] The results are shown in Figure 5. It shows that fibroblasts from different donor sources consistently maintain high levels of TGF-β secretion, which does not change significantly with cell passage. TGF-β can inhibit the proliferation and differentiation of Th1, Th2, and CTLs, thereby suppressing local inflammatory responses. It can inhibit the proliferation of PBMCs in cell-based experiments and treat knee osteoarthritis in animal studies.

[0075] 4. Secretion of human stromal cell-derived factor (CXCL12) by fibroblasts

[0076] 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.

[0077] The results are shown in Figure 6, which shows that fibroblasts from different donor sources can maintain high levels of CXCL12 secretion, and this does not change significantly with cell passage. Although low concentrations of CXCL12 can recruit immune cells, high concentrations can repel CD4+ and CD8+ T cells, inhibiting their local accumulation and alleviating local immune responses. In cell-based experiments, it can inhibit the proliferation of PBMCs and, in animal experiments, treat knee osteoarthritis.

[0078] Example 4: Application of fibroblasts

[0079] 1. Osteoarthritis modeling method

[0080] Select the male SD rat of 9-10 week size of age in week, body weight at 250-300g as research object, by rat with after isoflurane anesthesia, knee joint shaving, 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, by anterior cruciate ligament of knee joint transverse section.Then sew up muscle and skin, after iodine swab disinfection, be positioned over heating pad and rewarm, treat that it is put back into cage box and raise after waking up.It is model group that the knee joint of rat carries out cruciate ligament transverse section group, and the knee joint that is not processed is normal group, and it is experimental group that knee joint carries out cruciate ligament transverse section and is injected into the fibroblast group that embodiment two obtains.

[0081] 2. Cell Injection

[0082] Two weeks after surgery, the test substance (fibroblasts) was removed from 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, shaking it from side to side. All the liquid was removed with a pipette and added to a 50 mL sterile centrifuge tube. The centrifuge was set at 200g for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were diluted to 2E+07 / mL with normal saline. Using a microinjection needle, 1E+06 cells (50 μL) were injected into the joint cavity. The knee joint injected with the test substance served as the experimental group.

[0083] 3. Data Collection

[0084] The rats were euthanized with CO2 28 days after the first treatment, and the right and left knee joints were removed by dissection and sent to a third party for HE staining.

[0085] 4. Experimental results

[0086] As shown in Figures 7A to 7F, Figures 7A and 7D are staining images of the left and right knee joint tissues of the normal group, respectively. The joints are relatively smooth and without damage. Figures 7B and 7E are staining images of the left and right knee joint tissues of the experimental group, respectively. There are damages at the joints. However, compared with the modeling groups in Figures 7C and 7F, the joint damages in the experimental group injected with fibroblasts were better repaired. Fibroblasts can better repair the knee vallecula arthritis caused by ligament transection.

[0087] 5. Acute-on-chronic liver failure modeling method

[0088] 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 rat peritoneum at 1 mg / kg. The modeling group did not receive treatment. After the LPS injection, the experimental group was injected with the fibroblasts obtained in Example 2.

[0089] 6. Cell Injection

[0090] Remove the test substance (fibroblasts) from the liquid nitrogen tank and transport them on dry ice. Immediately immerse the test substance in a 37°C water bath and gently shake until the liquid is completely melted, shaking it side to side. Remove all the liquid with a pipette and add it to a 50mL sterile centrifuge tube. Centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant and dilute the cells to 2E+07 / mL with saline. Anesthetize the rat with isoflurane and secure it in the supine position on a surgical board while anesthesia is maintained. Isolate the portal vein and inject 1E+07 cells (1000 μL) into the portal vein using a syringe.

[0091] 7. Data Collection

[0092] The rats were euthanized with CO2 3 days after the first treatment, and the liver tissues were taken out after dissection and sent to a third party for HE staining, and the blood was taken for transaminase detection.

[0093] 8. Experimental Results

[0094] As shown in Figures 8A-8F, the normal group consisted of male rats, the model group consisted of male rats modeled with CCL4 and LPS, and the experimental group consisted of fibroblast-injected rats modeled with CCL4 and LPS. Figure 8A shows the results of alanine aminotransferase detection after blood collection. The alanine aminotransferase in the model group was significantly elevated, while that in the experimental group was significantly decreased, approaching the values ​​of the normal group. Figure 8B shows the results of aspartate aminotransferase detection after blood collection. The aspartate aminotransferase in the model group was significantly elevated, while that in the experimental group was significantly decreased, approaching the values ​​of the normal group. Figure 8C shows the survival curves of the three groups of animals, with 10 animals in each group as the starting number. No animals died in the normal group, 5 animals died in the model group, and 2 animals died in the experimental group. Figure 8D shows the results of pathological staining of the livers of the animals in the normal group. Figure 8E shows the results of pathological staining of the livers of the animals in the model group. Figure 8F shows the results of pathological staining of the livers of the animals in the experimental group.

Claims

1. A method for preparing fibroblasts, comprising the following steps: S1: Primary fibroblasts were obtained by digesting and isolating foreskin tissue with Clostridium peptidase A. S2: culturing the primary fibroblasts in vitro using a serum-free medium; the serum-free medium is high-glucose DMEM supplemented with hydrocortisone, bFGF, and human platelet lysate; S3: The obtained fibroblasts have a positive expression rate of CD105, CD90, CD73, COL1A1, and FAP exceeding 80%, a negative expression rate of CD36, CD39, CD45, and an HLA class II molecule expression rate less than 5%, and can secrete TGF-β.

2. The method according to claim 1, wherein The foreskin tissue is the foreskin tissue discarded from clinical circumcision surgery.

3. The method according to claim 2, wherein 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 method according to claim 1, wherein The method of S1 is: S1-1: After washing and disinfecting the foreskin tissue with normal saline, neutral proteinase A was added, incubated, and then washed with pre-cooled normal saline. The dermis and epidermis of the tissue were mechanically separated, the epidermis was removed, and the dermis was minced as much as possible, washed with pre-cooled normal saline, and incubated with Clostridium peptidase A to obtain primary fibroblasts; S1-2: Use a 70-μm cell strainer to sieve the cell suspension obtained in S1-11 with the aid of physiological saline, collect the cell suspension under the cell strainer, centrifuge and discard the supernatant, wash by centrifugation with serum-free medium, and resuspend in serum-free medium to obtain a primary cell suspension; S1-3: Using a 40-μm cell strainer, the primary cell suspension is subjected to sieve sorting with the aid of physiological saline, and the cell suspension harvested by filtration under the cell strainer is collected to complete sieve sorting. The obtained filtrate is centrifuged and the supernatant is removed to obtain primary fibroblasts.

5. The method according to claim 4, wherein The method of S1 is: S1-1: After cleaning and disinfecting the foreskin tissue with normal saline, add neutral proteinase A at a final concentration of 2 DMC U / mL and incubate at 37°C for 16-20 hours. Rinse 1-2 times with pre-cooled normal saline. Use sterile forceps to separate the dermis and epidermis of the tissue, remove the epidermis, and cut the dermis into pieces as small as 0.5-2 mm. Rinse 1-2 times with pre-cooled normal saline, transfer to a 50 ml centrifuge tube, and add Clostridium peptidase A at a final concentration of 0.1 PZ U / mL and incubate at 37°C for 2-3 hours. S1-2: Using a 70-μm cell strainer, the cell suspension is screened with the aid of physiological saline, the cell suspension under the cell strainer is collected, centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and the cell suspension is washed 1-2 times by centrifugation with serum-free medium, and resuspended in serum-free medium to obtain a primary cell suspension; 6. The method according to claim 1, wherein The method of S2 is: 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 trypsin digestion solution, and then subcultured in a serum-free medium to obtain fibroblasts.

7. The method according to claim 6, wherein Method S2: The trypsin digestion solution is digested for 1-5 minutes; the concentration of hydrocortisone is 5-25 ng / mL, the concentration of bFGF is 5-30 ng / mL, and the human platelet lysate is 4-10%; preferably, the concentration of hydrocortisone is 10-20 ng / mL, the concentration of bFGF is 15-25 ng / mL, and the human platelet lysate is 6-9%.

8. Fibroblasts obtained by the method according to any one of claims 1 to 7, wherein the fibroblasts have a positive expression rate of more than 80% for CD105, CD90, CD73, COL1A1, and FAP, a negative expression rate of less than 5% for CD36, CD39, CD45, and HLA class II molecules, and can secrete TGF-β.

9. Use of the fibroblasts according to claim 8 in preparing a medicament for treating an inflammatory disease, preferably, the inflammatory disease is local inflammation of osteoarthritis or acute liver failure.

10. The use according to claim 9, characterized in that: The diseases are osteoarthritis, acute liver failure.

11. A pharmaceutical composition comprising the fibroblasts according to claim 8 as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein It is in a dosage form suitable for injection.

13. The pharmaceutical composition according to claim 12, wherein The drug is suitable for intra-articular injection, intramuscular injection and intravenous injection.

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