Method for preparing recombinant chondroprogenitor cell, obtained recombinant chondroprogenitor cell and use

Abstract

Disclosed in the present application are a method for preparing a recombinant chondroprogenitor cell, the obtained recombinant chondroprogenitor cell, and the use in the field of genetic engineering. The technical problem to be solved by the present application is how to provide a cell product capable of treating osteoarthritis. In order to solve the technical problem, the present application provides a method for preparing a recombinant chondroprogenitor cell. The method comprises the following steps: 1) separating chondroprogenitor cells, wherein ex-vivo articular hyaline cartilage is enzymatically digested to obtain an enzymatically digested product, and the enzymatically digested product is cultured by means of the migration property of chondroprogenitor cells to obtain the chondroprogenitor cells; and 2) introducing a gene encoding TNFAIP3 protein into the chondroprogenitor cells to obtain the recombinant chondroprogenitor cells. The present application can be used for treating and / or preventing and / or alleviating and / or improving osteoarthritis.

Description

Method for preparing recombinant chondrocytes and obtained recombinant chondrocytes and their applications

[0001] Cross-reference to related applications:

[0002] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on September 13, 2023, with application number 202311178528.3 and invention name “Method for preparing recombinant chondrogenic progenitor cells and obtained recombinant chondrogenic progenitor cells and their application”. The entire contents of the patent application are hereby incorporated herein by reference. Technical Field

[0003] The present application relates to a method for preparing recombinant chondrocytes in the field of genetic engineering, and the obtained recombinant chondrocytes and their applications. Background Art

[0004] Osteoarthritis (OA) is a chronic degenerative disease characterized by destruction of joint cartilage and persistent pain, as well as structural and functional damage. OA has a high incidence, a long course, and an increasing prevalence year by year. Currently, drugs for the treatment of OA mainly relieve symptoms, and patients with mid- to late-stage OA require joint replacement surgery to relieve pain. Previous studies have shown that the occurrence and development of OA are related to multiple factors, including trauma, metabolism, and inflammation. These factors interact with each other, resulting in poor efficacy of most treatments. Therefore, there is an urgent need to develop new treatment strategies to delay the progression of OA.

[0005] In recent years, there has been considerable research on the role of stem cells in the treatment of osteoarthritis. However, due to the tissue heterogeneity of stem cells, their mechanism of action remains unclear, and their efficacy in treating osteoarthritis needs to be improved. Chondroprogenitor cells are tissue-specific stem cells with cartilage-forming potential and high replication capacity. Recent studies have shown that chondroprogenitor cells offer significant advantages in cartilage regeneration and repair, contributing to bone development, homeostasis, aging, and regeneration.

[0006] Invention Disclosure

[0007] The technical problem to be solved by this application is to provide a cell product capable of treating osteoarthritis. The technical problem to be solved is not limited to the technical subject described herein, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0008] To solve the above technical problems, this application provides the following technical solutions:

[0009] The present application provides a method for preparing recombinant chondrogenic cells, the method comprising the following steps:

[0010] 1) Isolation of chondrocyte progenitor cells: enzymatically digesting isolated hyaline cartilage to obtain an enzymatic hydrolysate, and culturing the enzymatic hydrolysate using the migration characteristics of chondrocyte progenitor cells to obtain chondrocyte progenitor cells;

[0011] 2) introducing a gene encoding tumor necrosis factor alpha-induced protein 3 into the chondrogenic progenitor cells to obtain the recombinant chondrogenic progenitor cells.

[0012] Furthermore, the recombinant chondrogenic cells in the above method express tumor necrosis factor alpha induced protein 3 (TNFAIP3).

[0013] The TNFAIP3 is A1), A2) or A3),

[0014] A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0015] A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID No. 1;

[0016] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0017] The above-mentioned identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0018] The aforementioned 80% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0019] The 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0020] The gene encoding the TNFAIP3 protein is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No. 2.

[0021] Furthermore, the above method does not include dedifferentiation of chondrocytes.

[0022] Furthermore, in the above method, culturing the enzymatic hydrolysate using the migration characteristics of chondrogenic cells includes the step of culturing using a culture medium for culturing adherent cells.

[0023] The culture medium for culturing adherent cells mentioned above may contain 10% by volume of FBS (fetal bovine serum).

[0024] The culture medium for culturing adherent cells described above can be a liquid culture medium (complete culture medium) obtained by adding FBS, penicillin and streptomycin to α-MEM culture medium, wherein the volume percentage of FBS in the culture medium for culturing adherent cells is 10%, the content of penicillin can be 100 units / ml, and the content of streptomycin can be 100 ng / ml.

[0025] Furthermore, the hyaline cartilage in the above method is derived from humans, specifically from human knee joints.

[0026] Furthermore, the enzymatic digestion in the above method is performed using collagenase.

[0027] The collagenase mentioned above may be type II collagenase.

[0028] The enzymatic digestion described above can be carried out in an enzymatic reaction system containing 125 CDU / mL of type II collagenase at 37° C. for 2 hours.

[0029] Furthermore, the chondroprogenitor cells in the above method have the ability to inhibit the formation of osteoclasts and / or bone resorption through secretion.

[0030] The present application also provides a recombinant chondroprogenitor cell prepared by the aforementioned method, wherein the recombinant chondroprogenitor cell expresses a protein having the amino acid sequence of SEQ ID No. 1.

[0031] The present application also provides a drug for treating and / or preventing and / or alleviating and / or improving osteoarthritis, which is composed of pharmaceutical excipients and the aforementioned recombinant chondrogenic cells.

[0032] Furthermore, the dosage form of the above-mentioned medicine is injection.

[0033] The present application also provides a use, namely, recombinant chondrocytes are used to improve traumatic osteoarthritis, wherein the recombinant chondrocytes express the tumor necrosis factor α-induced protein 3.

[0034] The recombinant chondrogenic cells are prepared by a method comprising the following steps:

[0035] 1) Isolation of chondrocyte progenitor cells: enzymatically digesting isolated hyaline cartilage to obtain an enzymatic hydrolysate, and culturing the enzymatic hydrolysate using the migration characteristics of chondrocyte progenitor cells to obtain chondrocyte progenitor cells;

[0036] 2) introducing a gene encoding tumor necrosis factor alpha-induced protein 3 into the chondrogenic progenitor cells to obtain the recombinant chondrogenic progenitor cells.

[0037] Furthermore, the recombinant chondrogenic cells in the above application express the tumor necrosis factor α-induced protein 3.

[0038] Furthermore, the use in improving traumatic osteoarthritis includes use in improving subchondral bone remodeling in traumatic osteoarthritis.

[0039] Furthermore, the method described in the above application does not include dedifferentiation of chondrocytes.

[0040] Furthermore, the hyaline cartilage in the above application is derived from humans.

[0041] Furthermore, the enzymatic digestion in the above application is performed using collagenase.

[0042] Furthermore, the dosage form of the drug in the above application is an injection.

[0043] The present invention also provides a method for improving subchondral bone remodeling or inhibiting necroptosis of subchondral osteoblasts in traumatic osteoarthritis, comprising administering the aforementioned recombinant chondroprogenitor cells or a drug containing the aforementioned recombinant chondroprogenitor cells to the bone joints of a subject to inhibit necroptosis of subchondral osteoblasts.

[0044] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human or non-human primate, such as a monkey or chimpanzee), a non-primate (e.g., a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, or whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a rat.

[0045] The present invention also provides any of the following uses of tumor necrosis factor alpha-induced protein 3 or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity and / or content of the protein:

[0046] B1) Drugs that improve subchondral bone remodeling in traumatic osteoarthritis;

[0047] B2) Drugs that inhibit necroptosis of subchondral osteoblasts;

[0048] The tumor necrosis factor alpha-induced protein 3 is any one of the following:

[0049] A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0050] A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A1) and having at least 80% identity with the protein and having the same function;

[0051] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in A1) or A2).

[0052] Furthermore, the regulation in the above-mentioned use may be upregulation, enhancement or increase of the expression of the gene encoding the aforementioned protein or the content or activity of the protein, and the regulation may also be downregulation, inhibition or reduction of the expression of the gene encoding the protein or the content or activity of the protein.

[0053] Furthermore, the substance in the above use is any one of the following:

[0054] B1), a nucleic acid molecule encoding the aforementioned protein;

[0055] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0056] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0057] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0058] B5) a transgenic animal cell line containing the nucleic acid molecule described in B1) or a transgenic animal cell line containing the expression cassette described in B2);

[0059] B6) transgenic animal tissue containing the nucleic acid molecule described in B1) or transgenic animal tissue containing the expression cassette described in B2);

[0060] B7) Transgenic animal organs containing the nucleic acid molecule described in B1) or transgenic animal organs containing the expression cassette described in B2).

[0061] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0062] Those skilled in the art can readily mutate the nucleotide sequence encoding the TNF-α-induced protein 3 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence of the isolated TNF-α-induced protein 3 protein of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the TNF-α-induced protein 3 protein and possess the function of the TNF-α-induced protein 3 protein.

[0063] The nucleotide sequence encoding the protein tumor necrosis factor alpha induced protein 3 of the present invention is SEQ ID No. 3, and the encoding gene encoding the protein tumor necrosis factor alpha induced protein 3 of the present invention is SEQ ID No. 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 shows fluorescence images of human chondrocytes modified with the TNFAIP3 gene and Western blotting results for TNFAIP3. The upper panel in Panel A shows a fluorescence image of chondrocytes modified with the TNFAIP3 overexpression vector LV5, while the lower panel in Panel A shows a fluorescence image of chondrocytes modified with the TNFAIP3 gene. Panel B shows Western blotting results of chondrocytes modified with both the LV5 and TNFAIP3 genes. The first lane on the left shows the LV5 vector, the second lane shows TNFAP3 overexpression, the first row shows Western blotting results with a TNFAP3 antibody, and the second row shows Western blotting results with a GAPDH antibody.

[0065] FIG2 is a growth curve diagram of chondrogenic cells of generations P0-P5 and a population doubling time diagram of generations P1-P5 provided in Example 1 of the present application.

[0066] FIG3 is a morphological diagram of chondrogenic cells provided in Example 1 of the present application.

[0067] FIG4 is a flow cytometry phenotype diagram of chondrogenic cells provided in Example 1 of the present application.

[0068] Figure 5 is a graph showing the identification of the three-lineage differentiation ability of chondrogenic cells provided in Example 1 of the present application. From left to right are the crystal violet staining results, ALP staining results, toluidine blue staining results, and Oil Red O staining results.

[0069] FIG6 is a diagram of karyotype analysis of chondrocyte progenitor cells provided in Example 1 of the present application.

[0070] FIG7 is a Masson staining image of chondroprogenitor cell tumorigenesis in vivo provided in Example 1 of the present application.

[0071] Figure 8 shows the bone imaging structure of the animal model of traumatic osteoarthritis improved by TNFAIP3 gene-modified human chondrocyte progenitor cells. The upper picture in the first column from left to right is a Micro-CT image of the joint of the control rat, the middle picture in the first column is an image of the subchondral bone corresponding to the joint of the control rat, and the lower picture in the first column is a three-dimensional image of the tibial plateau of the joint of the control rat; the upper picture in the second column is a Micro-CT image of the joint of the OA group rat, the middle picture in the second column is an image of the subchondral bone corresponding to the joint of the OA group rat, and the lower picture in the second column is a three-dimensional image of the tibial plateau of the joint of the OA group rat; the upper picture in the third column is a Micro-CT image of the joint of the rat in the group of human chondrocyte progenitor cells stably transfected with empty plasmids, and the middle ... The images of the subchondral bone corresponding to the joints of rats in the group of human chondrocytes transfected with empty plasmids, the lower image in the third column is a three-dimensional image of the tibial plateau of the joints of rats in the group of human chondrocytes stably transfected with empty plasmids injected into the joint cavity; the upper image in the fourth column is a Micro-CT image of the joints of rats in the group of chondrocytes modified with TNFAIP3 gene injected into the joint cavity, the middle image in the fourth column is an image of the subchondral bone corresponding to the joints of rats in the group of chondrocytes modified with TNFAIP3 gene injected into the joint cavity, and the lower image in the fourth column is a three-dimensional image of the tibial plateau of the joints of rats in the group of chondrocytes modified with TNFAIP3 gene injected into the joint cavity.

[0072] Figure 9 shows the joint histological morphology of TNFAIP3 gene-modified human chondrocyte progenitor cells in improving traumatic osteoarthritis. The first picture in the first column from left to right is the hematoxylin-eosin staining of the joints of the control group rats, the second picture in the first column is the safranin O fast green staining of the joints of the control group rats, the third picture in the first column is the toluidine blue staining of the joints of the control group rats, and the fourth picture in the first column is the immunohistochemistry of type I collagen in the joints of the control group rats; the first picture in the second column is the hematoxylin-eosin staining of the joints of the OA group rats, the second picture in the second column is the safranin O fast green staining of the joints of the OA group rats, the third picture in the second column is the toluidine blue staining of the joints of the OA group rats, and the fourth picture in the second column is the immunohistochemistry of type I collagen in the joints of the OA group rats; the first picture in the third column is the hematoxylin-eosin staining of the joints of the rats in the group of human chondrocyte progenitor cells stably transfected with an empty vector plasmid injected into the joint cavity, and the second picture in the third column is the immunohistochemistry of type I collagen in the joints of the rats in the group of human chondrocyte progenitor cells stably transfected with an empty vector plasmid injected into the joint cavity. Safranin O fast green staining of the joints of the rats in the cell group, the third figure in the third column is toluidine blue staining of the joints of the rats in the human chondroprogenitor cell group injected with stably transfected empty plasmids through the joint cavity, and the fourth figure in the third column is immunohistochemistry of type I collagen in the joints of the rats in the human chondroprogenitor cell group injected with stably transfected empty plasmids through the joint cavity; the first figure in the fourth column is hematoxylin-eosin staining of the joints of the rats in the chondroprogenitor cell group injected with TNFAIP3 gene-modified through the joint cavity, the second figure in the fourth column is Safranin O fast green staining of the joints of the rats in the chondroprogenitor cell group injected with TNFAIP3 gene-modified through the joint cavity, the third figure in the fourth column is toluidine blue staining of the joints of the rats in the chondroprogenitor cell group injected with TNFAIP3 gene-modified through the joint cavity, and the fourth figure in the fourth column is immunohistochemistry of type I collagen in the joints of the rats in the chondroprogenitor cell group injected with TNFAIP3 gene-modified through the joint cavity.

[0073] Figure 10 shows immunofluorescence staining of osteoblasts induced by necroptosis after treatment with culture supernatant of TNFAIP3 gene-modified human chondroprogenitor cells. The upper picture in the first column from left to right is the immunofluorescence of RIPK3 in subchondral osteoblasts of the control group, and the lower picture in the first column is the immunofluorescence of pMLKL in subchondral osteoblasts of the control group; the upper picture in the second column is the immunofluorescence of RIPK3 in subchondral osteoblasts of the necroptosis-induced group, and the lower picture in the second column is the immunofluorescence of pMLKL in subchondral osteoblasts of the necroptosis-induced group; the upper picture in the third column is the immunofluorescence of RIPK3 in subchondral osteoblasts of the necroptosis-induced group, and the lower picture in the second column is the immunofluorescence of pMLKL in subchondral osteoblasts of the necroptosis-induced group; the upper picture in the third column is the immunofluorescence of RIPK3 in subchondral osteoblasts of the necroptosis-induced group treated with culture medium containing 10% supernatant of human chondroprogenitor cells modified with stable empty vector plasmid. Immunofluorescence of PK3, the lower figure in the third column is the immunofluorescence of pMLKL in subchondral osteoblasts treated with a culture medium containing 10% supernatant of stably transfected empty plasmid human chondroprogenitor cells; the upper figure in the fourth column is the immunofluorescence of RIPK3 in subchondral osteoblasts treated with a culture medium containing 10% supernatant of chondroprogenitor cells modified with TNFAIP3 gene, and the lower figure in the fourth column is the immunofluorescence of pMLKL in subchondral osteoblasts treated with a culture medium containing 10% supernatant of chondroprogenitor cells modified with TNFAIP3 gene.

[0074] Figure 11 shows that intra-articular injection of TNFAIP3 gene-modified human chondrocyte progenitor cells inhibits necroptosis of subchondral osteoblasts. The first picture in the first column from left to right is the immunofluorescence of RIPK3 and OCN in the subchondral bone of the control rats, the second picture in the first column is a partial enlargement of the first picture, the third picture in the first column is the immunofluorescence of pMLKL and OCN in the subchondral bone of the control rats, and the fourth picture in the first column is a partial enlargement of the third picture; the first picture in the second column is the immunofluorescence of RIPK3 and OCN in the subchondral bone of the OA group rats, the second picture in the second column is a partial enlargement of the first picture, the third picture in the second column is the immunofluorescence of pMLKL and OCN in the subchondral bone of the OA group rats, and the fourth picture in the second column is a partial enlargement of the third picture; the first picture in the third column is the immunofluorescence of RIPK3 and OCN in the subchondral bone of the OA group rats injected with stably expressed empty plasmid human chondrocyte progenitor cells. The second figure in the third column is a partial enlarged view of the first figure. The third figure in the third column is the immunofluorescence of pMLKL and OCN in the subchondral bone of rats in the group of human chondroprogenitor cells stably transfected with empty plasmids injected into the joint cavity, and the fourth figure in the third column is a partial enlarged view of the third figure. The first figure in the fourth column is the immunofluorescence of RIPK3 and OCN in the subchondral bone of rats in the group of chondroprogenitor cells modified by TNFAIP3 gene injected into the joint cavity, the second figure in the fourth column is a partial enlarged view of the first figure. The third figure in the fourth column is the immunofluorescence of pMLKL and OCN in the subchondral bone of rats in the group of chondroprogenitor cells modified by TNFAIP3 gene injected into the joint cavity, and the fourth figure in the fourth column is a partial enlarged view of the third figure.

[0075] Best Mode for Carrying Out the Invention

[0076] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0077] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Definitions:

[0078] Lentiviral vector: a type of recombinant retroviral vector that can effectively integrate exogenous genes into host chromosomes, thereby achieving persistent expression.

[0079] The following examples are based on an internationally accepted third-generation vector system, modified to create a four-plasmid system. The transfer vector contains the lentiviral backbone for transferring the target gene and all cis-acting elements required for packaging and producing the corresponding genomic RNA. It can express the transferred gene or shRNA stably or conditionally inducibly, either singly or in multiple combinations. Furthermore, three helper plasmids provide the trans-acting factors required for viral packaging. A "self-inactivating" modification prevents progeny virus replication and transfer, ensuring the biosafety of the resulting lentivirus.

[0080] Target cells: refers to cells that become the target of attack by other cells, antibodies, viruses, drugs or other physiological substances, etc. In this application, it refers to the recipient chondrocyte progenitor cells that become the target of lentiviral attack.

[0081] Human knee tibial plateau tissue: tibial plateau obtained from knee arthroplasty.

[0082] Lamellar cartilage: A thin layer of cartilage cut from the hyaline cartilage layer, in the form of a sheet.

[0083] Hyaline cartilage is a translucent, strong connective tissue that provides structural support and a smooth joint surface. In this application, hyaline cartilage refers to the cartilage lining the tibial plateau of the knee joint. Damage or degeneration of hyaline cartilage can lead to joint pain and dysfunction, contributing to conditions such as osteoarthritis.

[0084] Example 1. Preparation of TNFAIP3 Gene-Modified Human Chondrogenic Cells and Detection of TNFAIP3 Expression

[0085] 1. Construction of TNFAIP3 overexpression vector

[0086] 1. Preparation of overexpression vector

[0087] The lentiviral plasmid containing the target gene TNFAIP3 is obtained by replacing the nucleotide sequence between the NotI and Bam HI enzyme recognition sites of the LV5 (EF-1a / GFP&Puro) vector (a product of GenePharma, recorded in the non-patent document "Hansen Yang, Jia Wang, Zheng Zhang, Rui Peng, Dan Lv, Handeng Liu, Yan Sun. Sp1-Induced lncRNA Rmrp Promotes Mesangial Cell Proliferation and Fibrosis in Diabetic Nephropathy by Modulating the miR-1a-3p / JunD Pathway. Front Endocrinol (Lausanne). 2021 Aug 27; 12: 690784. doi: 10.3389 / fendo.2021.690784.eCollection 2021.") with the DNA molecule shown in SEQ ID No. 2, and keeping the other nucleotide sequences unchanged. The lentiviral plasmid containing the target gene TNFAIP3 can express TNFAIP3 under the joint action of three auxiliary plasmids of the lentiviral packaging plasmid.

[0088] Amino acid sequence of TNFAIP3 (SEQ ID No. 1)

[0089] TNFAIP3 coding region sequence (SEQ ID No. 2)

[0090] Nucleotide sequence of TNFAIP3 (SEQ ID No. 3)

[0091] 2. Preparation of lentivirus expressing TNFAIP3

[0092] Three auxiliary packaging element vector plasmids, pGag / Pol plasmid, pRev plasmid and pVSV-G plasmid (GenePharma) and the recombinant overexpression vector of TNFAIP3 were co-transfected into 293T cells according to the operation manual of the recombinant lentivirus of GenePharma. The cell culture supernatant was collected 72 hours after transfection to obtain the lentivirus expressing TNFAIP3 mRNA.

[0093] Preparation of lentivirus containing empty plasmid: replace the TNFAIP3 overexpression plasmid with the LV5 (EF-1a / GFP&Puro) vector empty plasmid. The rest of the operation is the same as the operation steps of the lentivirus expressing TNFAIP3.

[0094] 3. Perform lentiviral cell transfection

[0095] 1) Plate recipient chondrogenic cells onto a six-well plate and observe cell growth density under a microscope. Infect cells with viruses when the cell density reaches approximately 30%. Add the appropriate volume of TNFAIP3-expressing lentivirus and empty vector-containing lentivirus, respectively, based on the MOI and virus titer. Calculate the volume of virus using the following formula: (MOI x number of cells) / virus titer. Prepare the infection system: 1 ml complete medium + 1 μl transfection agent + the volume of virus. Incubate at 37°C for 72 hours, then replace with complete medium and continue incubation at 37°C, 5% CO2.

[0096] 2) Three days after transfection, observe the expression of green fluorescent protein in the cells using a fluorescence microscope. After observation, add puromycin to the six-well plate to screen the virus-infected cells for resistance. The final puromycin concentration in each well of the six-well plate is 1 μg / ml. Use uninfected cells as a control. Screening ends when the control cells are completely dead.

[0097] 3) Continue to culture the cells that have not died in 2) and perform regular subculture on them, and finally subculture them to 75cm 2 Cell culture flask.

[0098] 4) Since TNFAIP3 mRNA transcribed by the lentivirus expressing TNFAIP3 mRNA in recipient chondrocytes can be translated into TNFAIP3 protein in the cytoplasm of recipient chondrocytes, TNFAIP3 protein expression can be detected by Western blotting. The TNFAIP3 primary antibody used in Western blotting was from Proteintech (66695-1-Ig), and the internal control GAPDH was from Proteintech (10494-1-AP).

[0099] The left image of Figure 1 is a fluorescence image of human chondrocytes modified with the TNFAIP3 gene. As can be seen from the left image of Figure 1, human chondrocytes modified with the TNFAIP3 lentivirus and the empty plasmid clearly express green fluorescence. The right image of Figure 1 is the Western blotting expression result of TNFAIP3. The right image of Figure 1 shows that the expression level of TNFAIP3 in human chondrocytes modified with the TNFAIP3 overexpression gene is significantly higher than the expression level of TNFAIP3 in human chondrocytes stably transfected with the empty plasmid. This shows that the TNFAIP3 overexpression construct was successful. The cells that were verified to be correct were named human chondrocytes modified with the TNFAIP3 overexpression gene. The lentivirus containing the empty plasmid was transferred into human chondrocytes according to the above method, and the cells that were verified to be correct were named human chondrocytes stably transfected with the empty plasmid.

[0100] The recipient chondrocytes are P3 chondrocytes, which are prepared according to the following method:

[0101] 1. Acquisition of joint platform tissue

[0102] Discarded human knee tibial plateau tissues were collected from the hospital (the patients had signed informed consent).

[0103] 2. Isolation and Culture of Chondrogenic Cells

[0104] The following operations were performed under sterile conditions.

[0105] 1. Transfer the collected human knee tibial plateau tissue to a sterile collection bottle and store at 2-8°C. Wash the tissue for 5 minutes in a clean bench with pre-chilled PBS phosphate buffered saline containing 1% double-antibody (100 units / ml penicillin and 100 ng / ml streptomycin).

[0106] 2) Use a sterile scalpel to dissect hyaline cartilage from the middle region of the tibial plateau on the opposite side of the normal human knee joint. First, use the scalpel to remove a thin sheet of cartilage, slicing from the central, weight-bearing surface of the joint, avoiding the edges. This hyaline cartilage is then placed in a six-well plate. The joint surface is hyaline cartilage, rich in stem cells, while the edges of the joint are more fibrocartilage-rich.

[0107] The relatively normal side refers to the side with relatively mild joint wear symptoms (pathological grade: S1-S2).

[0108] 3). Use ophthalmic scissors to cut the hyaline cartilage into pieces to obtain shredded hyaline cartilage.

[0109] 4). Transfer the minced hyaline cartilage to a 10 mL centrifuge tube, wash three times with PBS containing 1% double antibody, and remove the supernatant.

[0110] 5). Preheat 0.1% type II collagenase (Sigma, catalog number V900892) solution at 37°C, add 2 times the volume of hyaline cartilage tissue to the centrifuge tube in step 4) to obtain an enzymatic reaction system. The content of type II collagenase in the enzymatic digestion reaction system is 125 CDU / mL. After vortex mixing, place the mixture in a shaker at 37°C at 80 rpm for 2 hours to obtain digested cartilage tissue.

[0111] 6) Centrifuge the cartilage tissue after enzymatic digestion in step 5), aspirate the collagenase, add 5 mL of PBS containing 1% double antibody to the centrifuge tube and resuspend, centrifuge at 500 rpm for 3 minutes, discard the supernatant, and take the precipitate to obtain the cleaned cartilage tissue block (i.e., enzymatic hydrolysate).

[0112] 7). Culture and passage of chondrogenic cells

[0113] Culture medium for adherent cells: α-MEM (Gibco, Catalog No. C12571500BT) supplemented with FBS, penicillin, and streptomycin (also called complete medium). For adherent cells, the volume percentage of FBS is 10%, the penicillin content is 100 units / ml, and the streptomycin content is 100 ng / ml.

[0114] ① Transfer the cartilage tissue block cleaned in step 6) to a 25cm 2 About 5 mL of culture medium for culturing adherent cells was added to the culture flask, and the flask was moved to an incubator and cultured for 3 days without moving. The cells were designated as P0 generation chondrogenic progenitor cells (P0 for short).

[0115] ② When the P0 chondrogenic cells grow to a fusion rate of 80%-90%, remove the culture medium in the culture flask and add 0.25% EDTA-trypsin (Servicebio, product number G4004-100ML) for digestion. When the cells become round, add 2 times the volume of the above complete medium to terminate the digestion. After collection and centrifugation, resuspend with an appropriate amount of complete medium to adjust the cell density to 5×10 5 Cells were seeded per flask and cultured in a 37°C, 5% CO2 incubator. The medium was replaced every three days with 10% FBS-containing α-MEM medium to obtain P1 chondrogenic cells (abbreviated as P1). 10% FBS-containing α-MEM medium is a liquid culture medium obtained by adding FBS to α-MEM medium, with the volume content of FBS in the liquid culture medium being 10%.

[0116] ③ According to the method shown in step ②, the P1 generation chondrocytes were subcultured to the P3 generation, and named as the P3 generation chondrocytes (abbreviated as P3). After culturing the P3 generation chondrocytes according to the above method, the P4 generation chondrocytes (abbreviated as P4), the P5 generation chondrocytes (abbreviated as P5) and the P8 generation chondrocytes (abbreviated as P8) were collected. The left figure in Figure 2 is the growth curve of the P0-P5 generation chondrocytes, and the right figure in Figure 2 is the population doubling time (PDT) of the P1-P5 generation chondrocytes. (PDT = t × [lg2 / (lgNt-lgN0)]), Nt is the terminal number of cultured cells at time t, t is the 5th day (P1), 6.5th day (P2), 9th day (P3), 12th day (P4) or 16th day (P5), N0 is the initial number of cultured cells, and the initial number is 2×10 5 cells, that is, the number of cells seeded per well.

[0117] 8) Identify the cells in step 2:

[0118] ①Morphological identification

[0119] Using an optical microscope, we observed and photographed chondrocytes at passages P0, P4, and P8. The results showed that the chondrocytes were spindle-shaped and grew in a fibroblast-like pattern (Figure 3).

[0120] ②Flow cytometry

[0121] In the flow cytometry tube, adjust the concentration of P3 chondrogenic progenitor cells to 5×10 5100 μL cells / tube, and the final volume was 100 μL. 5 μL of CD45 flow cytometry antibody (Biolegend, catalog number 304016), CD31 flow cytometry antibody (Biolegend, catalog number 303118), CD235A flow cytometry antibody (Biolegend, catalog number 349112), CD29 flow cytometry antibody (eBioscience, catalog number 12-0299-41), CD44 flow cytometry antibody (Invitrogen, catalog number 17-0441-81), CD90 flow cytometry antibody (Biolegend, catalog number 328108), CD105 flow cytometry antibody (Invitrogen, catalog number 1930337), and PDPN flow cytometry antibody (Biolegend, catalog number 337022) were added to each flow cytometry tube, incubated at 4°C in the dark for 30 min, washed three times with PBS, and analyzed by flow cytometry. Figure 4 is the result of flow cytometry detection. It can be seen from Figure 4 that the isolated P3 generation chondrocytes do not express surface markers of hematopoietic cells, endothelial cells and erythrocytes such as CD45, CD31 and CD235A, but express chondrocyte markers CD29, CD44, CD90, CD105 and PDPN (podoplanin).

[0122] ③ Identification of self-renewal and trilineage differentiation abilities

[0123] A. Self-renewal capacity of chondrocyte progenitor cells

[0124] P3 chondrogenic cells were plated at 500 cells / well, and 2 mL of 10% FBS α-MEM medium was added. The cells were left undisturbed for the first three days; then the complete medium was replaced every 3 days; after 10 days, the cells were fixed and stained with 0.1% crystal violet solution.

[0125] The first image from the left in FIG5 is crystal violet staining, in which the presence of purple staining indicates the formation of cell colonies.

[0126] B. Induction of chondrogenic progenitor cells into osteoblasts

[0127] Experimental group: P3 chondrogenic cells were cultured at 5×10 3 Cells were seeded per well in a 48-well plate, and osteogenic induction medium (purchased from Cyagen, catalog number HUXMX-90021) was replaced every 3 days. After 10 days of induction, alkaline phosphatase (ALP) staining was performed.

[0128] Control group: The osteogenic induction medium was replaced with α-MEM medium containing 10% FBS, and other operations were the same as those of the experimental group.

[0129] The second picture from the left in Figure 5 is ALP staining, in which the presence of red staining indicates the presence of osteoblasts, that is, P3 chondroprogenitor cells can be induced to differentiate into osteoblasts.

[0130] C. Induction of cartilage progenitor cells to differentiate into chondrocytes

[0131] Experimental group: P3 chondrogenic cells were cultured at 5×10 3 Cells were seeded per well in a 48-well plate and replaced with chondrogenic induction medium (purchased from Cyagen, catalog number HUXMX-90041) every 3 days. After 14 days of induction, cells were stained with toluidine blue.

[0132] Control group: α-MEM medium containing 10% FBS was replaced with chondrogenic induction medium, and other operations were the same as those of the experimental group.

[0133] The third picture from the left in Figure 5 is Toluidine Blue staining, in which the presence of purple staining indicates the presence of chondroblasts, that is, P3 chondroprogenitor cells can be induced to differentiate into chondroblasts.

[0134] D. Induction of chondrocyte progenitor cells to differentiate into adipocytes

[0135] Experimental group: P3 chondrogenic cells were cultured at a rate of 1×10 4 Cells were seeded in 48-well plates at 100 cells / well and replaced with adipogenic induction medium (purchased from Cyagen, catalog number HUXMX-90031) every 3 days. After 14 days of induction, cells were stained with Oil Red O.

[0136] Control group: The adipogenic induction medium was replaced with α-MEM medium containing 10% FBS, and other operations were the same as those of the experimental group.

[0137] The fourth picture from the left in Figure 5 shows Oil Red O staining, in which the presence of red staining indicates the presence of adipocytes, that is, P3 chondroprogenitor cells can be induced to differentiate into adipocytes.

[0138] ④ Karyotype analysis

[0139] Before harvesting, treat P3 cell culture flasks with 20 μL of colchicine solution (20 μg / mL) for 20 minutes and harvest cells by centrifugation at 1100 rpm for 10 minutes. Add 0.075 M potassium chloride solution (solute: potassium chloride, solvent: water) and incubate in a 37°C waterbath for 15 minutes. Pre-fix with 1 mL of fresh fixative (acetic acid:methanol = 1:3). After centrifugation, retain an appropriate amount of fixative based on cell count. Place 100 μL of the cell suspension on a slide, air-dry, and bake in an oven at 90°C for 2 hours. The baked slides were trypsinized for 30 seconds, terminated with FBS, and finally stained with Giemsa solution for 2.5 minutes. Rinse with tap water, air-dry, and mount.

[0140] Slides were scanned using a GSL120 fully automated fluorescence microscope, and chromosome morphology was examined using Cytovision software. Twenty cells were examined per sample. Chromosome number, morphology, and structure were examined for each cell, and abnormal cells were classified and counted. Chromosome banding and karyotype nomenclature were based on the International System for Human Cytogenomic Nomenclature (ISCN 2020). The results showed that the isolated chondrogenic progenitor cells had normal chromosomes (Figure 6).

[0141] ⑤ Nude mouse subcutaneous tumor formation experiment

[0142] Experimental group: P3 chondrogenic progenitor cells were digested with trypsin and the cell concentration was adjusted to 5×10 6 8-week-old nude mice (purchased from Vital River, cat. no. 401) were subcutaneously injected with 0.2 ml of chondrocyte progenitor cells. Cell clump size was observed daily after injection. Four weeks later, cell clumps were harvested, fixed, paraffin-embedded, and sectioned for Masson staining.

[0143] Control group: Matrix gel was used instead of P3 chondrogenic progenitor cells, and other operations were the same as those in the experimental group.

[0144] The results showed that no abnormalities were observed at the injection site 4 weeks later, with no nested or glandular arrangement of cells, and no cell deformation or necrosis ( Figure 7 ).

[0145] Example 2: Experimental study on the treatment of osteoarthritis with human chondroprogenitor cells modified with TNFAIP3 gene

[0146] 1. Preparation of TNFAIP3 gene-modified human chondroprogenitor cell preparation

[0147] Preparation of human chondrocytes modified by overexpression of TNFAIP3: human chondrocytes modified by overexpression of TNFAIP3, which were in good growth condition and prepared in Example 1, were added with an appropriate volume of 0.25% trypsin and digested in a 37°C, 5% CO2 incubator for 3 min; 2 volumes of α-MEM medium containing 10% FBS were added to terminate the digestion, and the cells were centrifuged at 1200 rpm / min for 5 min. The concentration of human chondrocytes modified by overexpression of TNFAIP3 was adjusted to 10 with phosphate buffer. 6 cells / 100ul to obtain a TNFAIP3 overexpression gene-modified human chondroprogenitor cell preparation.

[0148] Preparation of human chondroprogenitor cells stably transfected with an empty plasmid: human chondroprogenitor cells stably transfected with an empty plasmid are used instead of human chondroprogenitor cells modified by the TNFAIP3 overexpression gene. Other operations are the same as those for the preparation of human chondroprogenitor cells modified by the TNFAIP3 overexpression gene.

[0149] 2. Construction of Traumatic Osteoarthritis Rat Model and Sham-Surgery Rat Model

[0150] (1) Sterilize the required surgical instruments in advance. Rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., strain code: 101) were anesthetized by intraperitoneal injection of sodium pentobarbital. After successful anesthesia, the hair around the knee joint was shaved clean, and then the rats were fixed on the operating table and iodine was wiped on the knee joint for sterilization. The rats for the osteoarthritis rat model were longitudinally incised from the inside of the patella with a surgical blade, the fascia was torn open, the anterior cruciate ligament was exposed, and it was cut with a scalpel; the patella was repositioned, and the anterior drawer test was performed to verify whether the model was successful; after the model was successfully established, the fascia and skin were sutured with surgical needles and thread, and the rats were returned to the cage after waking up. After one week of feeding, they were SD rats one week after modeling.

[0151] In the sham operation group (control group), only the joint capsule was opened without transecting the ligament, and then the incision was sutured.

[0152] (2) Experimental groups:

[0153] One week after modeling, SD rats were divided into three groups: OA group, vector LV5 group, and TNFAIP3 overexpression group, with 3 rats in each group; then intra-articular injection was performed. A control group was also set up, and the mice used in the control group were the sham operation group (control group) in step (1).

[0154] Each SD rat in the control group and OA group was injected with 0.1 mL of phosphate buffered saline into the joint cavity on the surgical side; each SD rat in the vector LV5 group was injected with 0.1 mL of human chondrocyte progenitor cell preparation stably transfected with empty plasmid (containing phosphate buffered saline and 1×10 6In the TNFAIP3 overexpression group, 0.1 mL of TNFAIP3 overexpression gene-modified human chondrocyte progenitor cell preparation (containing phosphate buffer and 1×10 6 TNFAIP3 overexpression gene-modified human chondrogenic progenitor cells).

[0155] 3. MicroCT system detects bone imaging structure

[0156] (1) Six weeks after intra-articular injection, the SD rats were euthanized, and the knee joints of the SD rats were scanned with microCT (Scanco Medical, Bassersodrf, Zurich, Switzerland) to detect bone imaging structures. The severity of osteoarthritis was evaluated based on the degree of bone hyperplasia and joint destruction.

[0157] (2) Results of bone imaging structure detection (Figure 8): The first column in Figure 8 shows, from top to bottom, the whole joint Micro CT, subchondral bone Micro CT, and tibia of the SD rats in the control group. It can be seen that the joint surface of the control group is flat and there is no obvious bone hyperplasia; the second column in Figure 8 shows, from top to bottom, the whole joint Micro CT, subchondral bone Micro CT, and tibia of the SD rats in the OA group. It can be seen that the joint surface of the OA group is obviously damaged, the subchondral bone is damaged, and the trabecular spacing is increased; the third column in Figure 8 shows, from top to bottom, the whole joint Micro CT, subchondral bone Micro CT, and tibia of the SD rats in the vector LV5 group. The fourth column in Figure 8 shows, from top to bottom, the whole joint Micro CT, subchondral bone Micro CT, and tibia of the SD rats in the TNFAIP3 overexpression group. It can be seen that compared with the vector LV5 group, TNFAIP3 gene-modified human chondroprogenitor cells alleviated the damage of the joint surface to a certain extent. The results showed that TNFAIP3 gene-modified human chondroprogenitor cells could improve bone imaging structure and reduce joint damage in a rat model of traumatic osteoarthritis.

[0158] 4. Tissue section pathology and immunohistochemical staining

[0159] (1) Six weeks after intra-articular injection, SD rats were euthanized, and knee joint tissues were fixed in paraformaldehyde and decalcified in 10% EDTA decalcification solution (solute: EDTA, solvent: neutral phosphate buffer) for 4 weeks. They were then paraffin-embedded and cut into 4 μm sections.

[0160] (2) The changes in tissue morphology were observed by staining with hematoxylin-eosin (H&E), safranin O-fast green (SOFG), and toluidine blue.

[0161] The results showed (Figure 9): Compared with the control group (Figures 1-3 from top to bottom in the first column of Figure 9), the OA group (Figures 1-3 from top to bottom in the second column of Figure 9) had an uneven joint surface, a thinner cartilage layer, and a reduced cartilage matrix; compared with the OA group and the vector LV5 group (Figures 1-3 from top to bottom in the fourth column of Figure 9), after treatment with TNFAIP3 gene-modified human chondroprogenitor cells (Figures 1-3 from top to bottom in the fourth column of Figure 9), the cartilage matrix increased, the joint surface was smoothed, and the cartilage tissue was protected to a certain extent.

[0162] (3) Immunohistochemical staining of collagen type I (COL I) was performed to analyze the expression of COL I in subchondral bone of joint tissue samples and evaluate subchondral bone remodeling. After quenching endogenous peroxidase and blocking nonspecific binding sites, the sections were incubated with anti-COL I (1:100; Servicebio, China) for immunohistochemical staining.

[0163] The experimental results (Figure 9) showed that compared with the control group (Figure 9, first column, fourth from top), the area of ​​COL I staining in the subchondral bone in the OA group (Figure 9, second column, fourth from top) was significantly reduced. Compared with the OA group and the vector LV5 group (Figure 9, third column, fourth from top), the area of ​​COL I staining in the subchondral bone increased after treatment with TNFAIP3 gene-modified human chondroprogenitor cells (Figure 9, fourth from top). These results suggest that TNFAIP3 gene-modified human chondroprogenitor cells can improve subchondral bone remodeling in traumatic osteoarthritis.

[0164] 5. Cell Immunofluorescence Staining

[0165] Culture medium containing TNF-α + ZVD-FMK: Add TNF-α and ZVD-FMK to α-MEM culture medium containing 10% FBS to make the TNF-α content reach 100 ng / mL and the ZVD-FMK content reach 40 mmol / L.

[0166] The culture medium containing 10% of the culture supernatant of human chondrocytes stably transfected with empty plasmid was inoculated with the human chondrocytes stably transfected with empty plasmid prepared in Example 1 at an appropriate density in a 25 cm 2The cells were cultured in a cell culture flask at 37°C and 5% CO2 in an incubator. When the cells grew to 50% confluence, the culture medium was replaced and the culture was continued for 48 hours to obtain the culture supernatant of human chondrocytes stably transfected with an empty plasmid. The culture supernatant of human chondrocytes stably transfected with an empty plasmid was mixed with the culture medium containing TNF-α+ZVD-FMK at a volume ratio of 1:9 to obtain a culture medium containing 10% of the culture supernatant of human chondrocytes stably transfected with an empty plasmid.

[0167] The culture medium containing 10% of the culture supernatant of TNFAIP3 gene-modified human chondroprogenitor cells was inoculated with the TNFAIP3 overexpression gene-modified human chondroprogenitor cells prepared in Example 1 at an appropriate density in a 25 cm 2 The cells were cultured in a cell culture flask at 37°C and 5% CO2 in a culture incubator. When the cells grew to 50% confluence, the culture medium was replaced and the culture was continued for 48 hours to obtain the culture supernatant of TNFAIP3-overexpressing gene-modified human chondrogenic progenitor cells. The culture supernatant of TNFAIP3-overexpressing gene-modified human chondrogenic progenitor cells was mixed with the culture medium containing TNF-α+ZVD-FMK at a volume ratio of 1:9 to obtain a culture medium containing 10% of the culture supernatant of TNFAIP3-overexpressing gene-modified human chondrogenic progenitor cells.

[0168] (1) The tibial plateau of patients undergoing total knee replacement was collected, bone tissue was separated from the subchondral bone and cut into 1-2 mm slices for culture. The culture medium was changed every three days. When osteoblasts migrated, grew and fused to 80%-90% confluence, they were stably passaged. Osteoblasts from passages P3-P6 were plated in six-well plates (2×10 5 cells / well), and after the cells adhered, the conditioned medium was replaced (the control group used complete medium, the necroptosis-inducing group used medium containing TNF-α+ZVD-FMK, the vector LV5 group used medium containing 10% culture supernatant of stably transfected empty plasmid human chondroprogenitor cells, and the TNFAIP3 overexpression group used medium containing 10% culture supernatant of TNFAIP3 gene-modified human chondroprogenitor cells).

[0169] (2) After 24 h of replacing the conditioned medium, osteoblasts were fixed and immunofluorescence staining was performed. The primary and secondary antibodies used were as follows: RIPK3 (1:300, Proteintech, 17563-1-AP), p-MLKL (1:300, Immunoway, YP1224), Alexa CY3-labeled goat anti-rabbit IgG (1:350, Servicebio, GB25303).

[0170] The results (Figure 10) showed increased RIPK3 and p-MLKL staining in osteoblasts in the induced group (Figure 10, second column) compared to the control group (Figure 10, first column). However, treatment with the culture supernatant of TNFAIP3-modified human chondroprogenitor cells (Figure 10, fourth column) showed decreased RIPK3 and p-MLKL staining in osteoblasts. These results suggest that the supernatant of TNFAIP3-modified human chondroprogenitor cells can partially inhibit apoptosis in induced subchondral osteoblasts.

[0171] 6. Immunofluorescence staining of tissue sections

[0172] Six weeks after intra-articular injection of SD rats, the rats were euthanized and the knee joint tissues were fixed in paraformaldehyde and decalcified in 10% EDTA decalcification solution (solvent: neutral phosphate buffer) for 4 weeks. They were then embedded in paraffin and cut into 4 μm sections. The primary and secondary antibodies used for immunofluorescence staining of tissue sections were as follows: RIPK3 (1:100, Proteintech, 17563-1-AP), p-MLKL (1:100, Immunoway, YP1224), OCN (1:100, Servicebio, GB11233), Alexa 488-labeled goat anti-rabbit IgG (1:200, Servicebio, GB25303), Alexa CY3-labeled goat anti-rabbit IgG (1:200, Servicebio, GB25303).

[0173] The results (Figure 11) showed that compared with the control group (four images in the first column of Figure 11), RIPK3 and p-MLKL staining in subchondral osteoblasts in the OA group (four images in the second column of Figure 11) increased; compared with the OA group and the vector LV5 group (four images in the third column of Figure 11), RIPK3 and p-MLKL staining in subchondral osteoblasts treated with TNFAIP3 gene-modified human chondroprogenitor cells (four images in the fourth column of Figure 11) decreased. These results suggest that TNFAIP3 gene-modified human chondroprogenitor cells can partially inhibit the necroptosis of subchondral osteoblasts in traumatic osteoarthritis and improve bone remodeling.

[0174] The above results indicate that TNFAIP3 gene-modified human chondroprogenitor cells can effectively alleviate the pathological damage of osteoarthritis, while inhibiting the necroptosis of subchondral osteoblasts, and have a protective effect on traumatic osteoarthritis.

[0175] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art.

[0176] Industrial Applications

[0177] This application demonstrates through experiments that TNFAIP3 gene-modified human chondroprogenitor cells can effectively alleviate the pathological damage of osteoarthritis and inhibit necroptosis of subchondral osteoblasts, demonstrating a protective effect against traumatic osteoarthritis. The present invention can be used to treat and / or prevent and / or alleviate and / or improve osteoarthritis.

Claims

1. A method for preparing recombinant chondrogenic cells, characterized in that: The method comprises the following steps: 1) Isolating chondrocyte progenitor cells: digesting isolated hyaline cartilage by enzymatic hydrolysis to obtain an enzymatic hydrolysate, and culturing the enzymatic hydrolysate by utilizing the migration characteristics of chondrocyte progenitor cells to obtain chondrocyte progenitor cells; 2) introducing a gene encoding tumor necrosis factor alpha-induced protein 3 into the chondrogenic cells to obtain the recombinant chondrogenic cells.

2. The method according to claim 1, characterized in that The recombinant chondrogenic cells express the tumor necrosis factor-α-induced protein 3.

3. The method according to claim 1, characterized in that The method does not involve dedifferentiation of chondrocytes.

4. The method according to claim 2, characterized in that: The method of culturing the enzymatic hydrolysate by utilizing the migration characteristics of chondrogenic cells comprises the step of culturing the enzymatic hydrolysate by utilizing a culture medium for culturing adherent cells.

5. The method according to claim 1, characterized in that The articular hyaline cartilage is derived from human.

6. The method according to any one of claims 1 to 5, characterized in that The enzymatic digestion is performed using collagenase.

7. The method according to any one of claims 1 to 5, characterized in that The chondroprogenitor cells have the ability to inhibit the formation of osteoclasts and / or bone resorption through secretion.

8. Recombinant chondrogenic cells, characterized in that The recombinant chondrogenic cells are prepared by the method according to any one of claims 1 to 6.

9. A drug for treating and / or preventing and / or alleviating and / or improving osteoarthritis, characterized in that: The medicine consists of pharmaceutical excipients and the recombinant chondrogenic cells according to claim 8.

10. The drug according to claim 9, characterized in that The dosage form of the drug is injection.

11. Use, characterized in that The use is to use the recombinant chondrocytes to improve traumatic osteoarthritis, and the recombinant chondrocytes express the tumor necrosis factor α-induced protein 3.

12. The use according to claim 11, characterized in that The improving of traumatic osteoarthritis includes improving subchondral bone remodeling in traumatic osteoarthritis.

13. The use according to claim 11, characterized in that The articular hyaline cartilage is derived from human.

14. The use according to any one of claims 11 to 13, characterized in that The enzymatic digestion is performed using collagenase.

15. A method for improving subchondral bone remodeling or inhibiting necroptosis of subchondral osteoblasts in traumatic osteoarthritis, comprising administering the recombinant chondroprogenitor cells of claim 1 or a drug containing the recombinant chondroprogenitor cells of claim 1 to a subject's bone joint to inhibit necroptosis of subchondral osteoblasts.

16. The method according to claim 15, characterized in that The subject suffers from osteoarthritis.

17. The method according to claim 15, characterized in that The subject suffers from traumatic osteoarthritis.

18. Use, characterized in that Any of the following uses of tumor necrosis factor alpha-induced protein 3 or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity and / or content of the protein: B1) for preparing a medicament for improving subchondral bone remodeling in traumatic osteoarthritis; B2) for preparing a drug for inhibiting necroptosis of subchondral osteoblasts; The tumor necrosis factor alpha-induced protein 3 is any one of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in A1) or A2).

19. The use according to claim 18, characterized in that The regulation may be up-regulation, enhancement or increase of the expression of the gene encoding the protein in claim 1 or the content or activity of the protein, or the regulation may be down-regulation, inhibition or reduction of the expression of the gene encoding the protein or the content or activity of the protein.

20. The use according to claim 18, characterized in that The substance is any of the following: B1), a nucleic acid molecule encoding the protein according to claim 18; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic animal cell line containing the nucleic acid molecule described in B1), or a transgenic animal cell line containing the expression cassette described in B2); B6), transgenic animal tissue containing the nucleic acid molecule described in B1), or transgenic animal tissue containing the expression cassette described in B2); B7), transgenic animal organs containing the nucleic acid molecule described in B1), or transgenic animal organs containing the expression cassette described in B2).

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