Use of substances increasing the content or activity of oskm in preventing aging of articular cartilage and treating osteoarthritic diseases

Abstract

The application discloses application of a substance with increased OSKM content or activity in preventing joint cartilage aging and treating osteoarthritis diseases. The OSKM of the application is obtained by connecting Oct4, Sox2, Klf4 and C-Myc. The experiment of the application proves that the OSKM or the substance with increased OSKM content or activity has a definite effect in preventing joint cartilage aging and treating osteoarthritis diseases, the application provides a new strategy for developing gene therapy for preventing joint cartilage aging and treating osteoarthritis diseases, and expands the selectable range of clinical gene therapy and drug therapy.

Description

Technical Field

[0001] This invention relates to the biomedical field and the application of substances that increase the content or activity of OSKM in preventing articular cartilage aging and treating osteoarthritis. Background Technology

[0002] Osteoarthritis is a chronic joint disease, more common in older adults. It is caused by cartilage wear and degeneration, leading to increased inflammation, compensatory osteophyte formation, and synovial hyperplasia, usually accompanied by joint pain and deformity. Treatment for osteoarthritis typically involves nonsteroidal anti-inflammatory drugs (NSAIDs) and weight loss, which can alleviate joint pain to some extent, but the long-term cure effect is limited. The surface of cartilage contains a group of cells with self-renewal capabilities (cartilage stem / progenitor cells). When cartilage tissue is damaged, these cells differentiate into chondrocytes for repair. During the development of osteoarthritis, senescence of these cells causes the rate of cartilage regeneration and repair to be less than the rate of degeneration, resulting in cartilage thinning and inducing arthritis. Gene therapy has been reported to introduce factors such as DGCR8, CBX4, YAP1, FOXD1, and CLOCK into the joint cavity, which can slow down the senescence of cartilage stem / progenitor cells, promote cartilage regeneration, and restore joint physiological function.

[0003] The four reprogramming factors OSKM (Oct4, Sox2, Klf4, C-Myc) can reprogram fibroblasts into induced pluripotent stem cells (iPS cells) in vitro. Intermittent expression of OSKM can alleviate aging in tissues such as the liver, muscle, kidney, and retina, and prolong the lifespan of mice. Currently, no research has shown the role of OSKM in the treatment or relief of osteoarthritis. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to treat and / or prevent osteoarthritis, or delay joint aging.

[0005] To address the aforementioned technical problems, this invention first provides the application of substances that reprogram four-factor OSKM or increase the content or activity of OSKM in the preparation of products for the treatment and / or prevention of osteoarthritis.

[0006] In the above applications, OSKM can be composed of Oct4, Sox2, Klf4 and C-Myc or connected by Oct4, Sox2, Klf4 and C-Myc.

[0007] In one embodiment of the present invention, OSKM is composed of Oct4, Sox2, Klf4 and C-Myc connected in sequence.

[0008] Oct4 can be the protein represented by positions 1-352 of SEQ ID No. 2 or positions 1-360 of SEQ ID No. 5;

[0009] Sox2 may be the protein represented by positions 379-697 of SEQ ID No. 2 or positions 387-703 of SEQ ID No. 5;

[0010] Klf4 may be the protein represented by positions 732-1205 of SEQ ID No. 2 or positions 738-1207 of SEQ ID No. 5;

[0011] C-Myc can be the protein represented by positions 1249-1687 of SEQ ID No. 2 or positions 1251-1689 of SEQ ID No. 5.

[0012] In SEQ ID No. 5, bits 1-360 represent OCT4, bits 387-703 represent SOX2, bits 738-1207 represent KLF4, and bits 1251-1689 represent C-MYC.

[0013] Specifically, OSKM can be A1), A2), or A3):

[0014] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0015] A2) A protein that has the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, but with one or more amino acid residues substituted and / or deleted and / or added;

[0016] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0017] To facilitate the purification of OSKM in A1), tags as shown in the table below can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0018] Table: Sequence of Labels

[0019] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0020] The OSKM protein in A2) above is a protein that shares 75% or more amino acid sequence identity with the protein shown in SEQ ID No. 2 and has the same function. The 75% or more identity refers to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. Specifically, the OSKM protein in A2) may be the protein shown in SEQ ID No. 5.

[0021] The OSKM protein in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0022] The gene encoding the OSKM protein in A2) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 4, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in the table above to its 5′ end and / or 3′ end. Specifically, the DNA molecule shown in SEQ ID No. 1 encodes the OSKM protein shown in SEQ ID No. 2, and the DNA molecule shown in SEQ ID No. 4 encodes the OSKM protein shown in SEQ ID No. 5.

[0023] In SEQ ID No. 4, bits 1-1080 represent OCT4, bits 1081-1158 represent P2A, bits 1159-2109 represent SOX2, bits 2110-2211 represent T2A, bits 2212-3621 represent KLF4, bits 3622-3750 represent E2A, and bits 3751-5070 represent C-MYC.

[0024] In the above applications, the substance that increases the content or activity of OSKM can be a substance that promotes the expression of the OSKM gene or a biological material related to OSKM.

[0025] The biomaterial is any one of B1) to B4) below:

[0026] B1) Nucleic acid molecules encoding OSKM;

[0027] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0029] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0030] In the above applications, the nucleic acid molecule described in B1) may be as follows: b11), b12), b13), or b14):

[0031] b11) The coding sequence is the DNA molecule of SEQ ID No. 1 in the sequence listing;

[0032] b12) The DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0033] b13) has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes a DNA molecule of OSKM;

[0034] b14) hybridizes under strict conditions with a nucleotide sequence defined by b11) or b12) or b13) and encodes a DNA molecule of OSKM.

[0035] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0036] Those skilled in the art can readily mutate the nucleotide sequence encoding the OSKM protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the OSKM protein isolated according to this invention, as long as they encode and function the OSKM protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0037] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2 of this invention. Identity can be evaluated visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Specifically, the DNA molecule described in b13) may be as shown in SEQ ID No. 4.

[0038] In the above applications, the stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 7% SDS, 0.5M NaPO4, and 1mM EDTA. Hybridization was performed in a mixed solution of NaPO4 and 1 mM EDTA, followed by rinsing at 65°C in 0.1×SSC and 0.1% SDS. Alternatively, hybridization was performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by rinsing once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS. Alternatively, hybridization was performed in a solution of 2×SSC and 0.1% SDS at 68°C, followed by rinsing twice for 5 min each time, and then hybridization was performed in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by rinsing twice for 15 min each time. Alternatively, hybridization was performed in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C, followed by rinsing.

[0039] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0040] In the above application, the expression cassette containing nucleic acid molecules (OSKM gene expression cassette) described in B2) refers to DNA capable of expressing the aforementioned OSKM protein in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for expressing any of the aforementioned proteins. The regulatory sequences, under compatible conditions, can guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence can be provided. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein into the secretion pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification. In these cases, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0041] Recombinant vectors containing the OSKM gene expression cassette can be constructed using existing expression vectors.

[0042] In the above applications, the vector can be a plasmid, granule, bacteriophage, or viral vector (such as a lentiviral vector).

[0043] B3) The recombinant vector may specifically be PLE4-OSKM. PLE4-OSKM is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the OSKM gene shown in SEQ ID No. 1 of the sequence listing. PLE4-OSKM can express the OSKM protein shown in SEQ ID No. 2.

[0044] In the above applications, the microorganisms may be yeast, bacteria, algae, or fungi.

[0045] In the above applications, the osteoarthritis may be age-related osteoarthritis or pathological osteoarthritis.

[0046] The treatment and / or prevention of osteoarthritis may be reflected in reducing the level of inflammation in the cartilage.

[0047] The present invention also provides the application of the reprogrammed four-factor OSKM or the substance that increases the content or activity of OSKM in the preparation of products that delay joint aging.

[0048] The joint aging mentioned may refer to the aging of articular cartilage.

[0049] The delay in joint aging can be manifested in improving the bioactivity of cartilage tissue cells, delaying articular cartilage aging, improving the mobility of aging animals, inhibiting bone hyperplasia, promoting cartilage tissue growth, inhibiting synovial hyperplasia, and / or increasing the number of cells that secrete the extracellular matrix necessary for cartilage.

[0050] The present invention also provides a product, wherein the active ingredient of the product is the reprogrammed four-factor OSKM or the substance that increases the content or activity of OSKM.

[0051] The product can be used to treat and / or prevent osteoarthritis and delay joint aging.

[0052] The experiments of this invention have confirmed that OSKM or substances that increase the content or activity of OSKM have a clear effect in preventing articular cartilage aging and treating osteoarthritis. This invention provides a new strategy for developing gene therapy for preventing articular cartilage aging and treating osteoarthritis, and expands the range of options for clinical gene therapy and drug therapy.

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0054] Figure 1To investigate the therapeutic effect of increasing OSKM levels on relieving osteoarthritis in aged mice. Figure (A) Schematic diagram evaluating the therapeutic effect of OSKM on osteoarthritis in aged mice. Figure (B) Real-time quantitative PCR (chain polymerase reaction) detection of transcriptional levels of Oct4, Sox2, Klf4, and C-Myc in articular cartilage of mice injected with PLE4-OSKM virus. Figure (C) Changes in grip strength of the hind limbs of mice after injection of PLE4-OSKM virus. Figure (D) CT scan to assess the volume of bone hyperplasia in the hind limb joints of aged mice after injection of PLE4-OSKM virus. Figure (E) Safranin-Fix-Green staining to assess the area of ​​articular cartilage in the hind limbs of aged mice after injection of PLE4-OSKM virus. Figure (F) H&E staining to assess the synovial hyperplasia in the hind limb joints of aged mice after injection of PLE4-OSKM virus. Figure (G) Immunohistochemical staining to detect the proportion of P21-positive cells in the articular cartilage of the hind limbs of aged mice after injection of PLE4-OSKM virus. Figure (H) shows the proportion of MMTV-positive cells in the hind limb articular cartilage of aged mice after PLE4-OSKM virus injection, detected by immunohistochemical staining. Figure (I) shows the proportion of IL-6-positive cells in the hind limb articular cartilage of aged mice after PLE4-OSKM virus injection, detected by immunohistochemical staining. Figure (J) shows the proportion of TNFα-positive cells in the hind limb articular cartilage of aged mice after PLE4-OSKM virus injection, detected by immunohistochemical staining. Figure (K) shows the proportion of Aggrecan-positive cells in the hind limb articular cartilage of aged mice after PLE4-OSKM virus injection, detected by immunohistochemical staining. Figure (L) shows the proportion of MMP13-positive cells in the hind limb articular cartilage of aged mice after PLE4-OSKM virus injection. The scale bars in Figure EL are 40 μm (top) and 10 μm (bottom).

[0055] Figure 2To investigate the therapeutic effect of increasing OSKM levels in alleviating ACLT osteoarthritis in a mouse model. Figure (A) Schematic diagram evaluating the therapeutic effect of OSKM on ACLT osteoarthritis in a mouse model. Figure (B) CT scan assessing the volume of bone hyperplasia in the hind limb joints of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (C) Safranin-Fix-Green staining assessing the OARSI score of the hind limb articular cartilage in ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (D) H&E staining assessing the synovial hyperplasia in the hind limb joints of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (E) Immunohistochemical staining detecting the proportion of Aggrecan-positive cells in the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (F) Immunohistochemical staining detecting the proportion of MMP13-positive cells in the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (G) shows the proportion of IL-6-positive cells in the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus, as detected by immunohistochemical staining. The scale bars in Figure CG are 40 μm (top) and 10 μm (bottom). Detailed Implementation

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques and conditions described in published literature in the field or according to the product instructions. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. All quantitative experiments in the following examples involved no fewer than 10 animals. All animal experiments were approved by the Animal Welfare Committee of the Institute of Zoology, Chinese Academy of Sciences. Experimental data are expressed as mean ± standard deviation and analyzed using GraghPad Prism 9 statistical software. A p-value < 0.05 was considered statistically significant between groups.

[0057] OSKM chimeric mice: documented in "Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, Li M, Lam D, Kurita M, Beyret E, Araoka T, Vazquez-Ferrer E, Donoso D, Roman JL, Xu J, Rodriguez Esteban C". G, The biological material is available to the public from the applicant and is intended solely for the purpose of repeating the relevant experiments of this invention and may not be used for any other purpose.

[0058] Human embryonic kidney 293T (HEK) cell line: ATCC product, catalog number CRL-3216.

[0059] HEK293T cell culture medium: 89% DMEM high glucose medium (HyClone), 10% fetal bovine serum (Gibco, 10100-147), 1% penicillin / streptomycin (Invitrogen, 15070-063).

[0060] Example 1: The therapeutic effect of increasing OSKM content on osteoarthritis in aged mice.

[0061] This embodiment demonstrates that injecting a lentiviral vector expressing OKSM into the joint cavity of aged mice can delay the aging of articular cartilage and alleviate osteoarthritis symptoms. The experimental steps are as follows:

[0062] 1. Preparation of lentivirus expressing mouse OSKM

[0063] 1.1 Preparation of recombinant vectors to enhance mouse OSKM gene

[0064] The template for amplifying the OSKM tandem expression sequence (Oct4-P2A-Sox2-T2A-Klf4-E2A-CMyc) was liver tissue DNA from OSKM chimeric mice, and the amplification primers used were as follows:

[0065] cDNA-OSKM-F: 5'-ATGGCTGGACACCTGGCTTCAGACTTCGCCTTCTC-3';

[0066] cDNA-OSKM-R: 5'-TTATGCACCAGAGTTTCGAAGCTGTTCGAGTTTGT-3'.

[0067] The full-length cDNA sequence of mouse OSKM, amplified by PCR (or directly synthesized, its sequence being SEQ ID No. 1 in the sequence listing, encoding the OSKM protein shown in SEQ ID No. 2), was ligated into the PLE4 vector, which was double-digested with BamHI (NEB) and MluI (NEB). The resulting recombinant vector with the correct sequence was denoted as recombinant vector PLE4-OSKM. PLE4-OSKM is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the OSKM gene shown in SEQ ID No. 1 in the sequence listing. PLE4-OSKM can express the OSKM protein shown in SEQ ID No. 2.

[0068] In SEQ ID No. 1, positions 1-1056 represent the Oct4 gene, positions 1057-1134 represent the P2A gene, positions 1135-2091 represent the Sox2 gene, positions 2092-2193 represent the T2A gene, positions 2194-3615 represent the Klf4 gene, positions 3616-3744 represent the E2A gene, and positions 3745-5061 represent the C-Myc gene. In SEQ ID No. 2, positions 1-352 represent Oct4, positions 379-697 represent Sox2, positions 732-1205 represent Klf4, and positions 1249-1687 represent C-Myc.

[0069] Following the method described above, a control recombinant vector (PLE4-GAL4) with the correct sequence was obtained. The primers used are as follows:

[0070] cDNA-GAL4-F: 5'-ATGAAGCTACTGTCTTCTATC-3';

[0071] cDNA-GAL4-R: 5'-CTAAACCTTTCTCTTTTTCTTGG-3'.

[0072] PLE4-GAL4 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the GAL4 gene shown in SEQ ID No. 3 of the sequence listing. PLE4-GAL4 can express the GAL4 protein.

[0073] 1.2 Preparation of PLE4-OSKM Lentiviral Concentrate

[0074] The PLE4-OSKM recombinant vector was co-transfected with the lentiviral packaging vectors psPAX2 (Addgene product, #12260) and pMD2.G (Addgene product, #12259) into 293T cells to obtain a lentivirus containing PLE4-OSKM. The steps are as follows:

[0075] 1.2.1 Using the Lipo3000 transfection kit (Thermo Scientific), 293T cells were co-transfected with the PLE4-OSKM recombinant vector, the lentiviral packaging vector psPAX2, and pMD2.G (ratio: 1 10cm dish of 293T cells: 9μg PLE4-OSKM recombinant vector, 6μg psPAX2, and 3μg pMD2.G), and incubated at 37°C.

[0076] 1.2.2 After culturing for 8 hours, replace with fresh 293T cell culture medium and continue culturing for 48-54 hours.

[0077] 1.2.3 Collect the culture medium supernatant, filter it through a 0.45 μm pore size filter membrane, and collect the filtrate.

[0078] 1.2.4 Centrifuge at 19,400 rpm for 2 hours at 4℃, discard the supernatant, and resuspend in PBS to obtain the concentrated lentivirus solution containing PLE4-OSKM, referred to as PLE4-OSKM lentivirus suspension.

[0079] Following the above method, PLE4-OSKM was replaced with PLE4-GAL4 to obtain a PLE4-GAL4 lentiviral suspension, which was then resuspended in PBS.

[0080] 2. Establish a naturally aging mouse model of osteoarthritis

[0081] Test mice: Male, 18-month-old SPF-grade mice (C57BL / 6J), from Spifort (Beijing) Biotechnology Co., Ltd. Mice housing conditions: 23℃, 12-hour light / 12-hour dark, free access to food and water. Experimental equipment and materials included: isoflurane gas anesthesia device, insulin injector.

[0082] Grouping: Mice were randomly divided into two groups of 11 each.

[0083] The PLE4-GAL4 group (11 mice) had 20 μl of PLE4-GAL4 lentivirus suspension injected into the left and right joint cavities of each mouse's hind limb.

[0084] The PLE4-OSKM group (11 mice) had 20 μl of PLE4-OSKM lentivirus suspension injected into the left and right joint cavities of each mouse's hind limb.

[0085] 3. Effectiveness Evaluation

[0086] 3.1 Supplementing with OSKM can delay joint aging, slow down the aging of articular cartilage, and improve arthritis symptoms.

[0087] Lentiviral suspension was injected into the hind limb joint cavity of aged mice, and the treatment effect was evaluated after 8 weeks. Figure 1 (A) The specific method is as follows:

[0088] 3.1.1 Behavioral evaluation of changes in joint mobility in mice

[0089] Eight weeks after injection of lentivirus suspension, the joint mobility of mice was evaluated by measuring the grip strength of their hind limbs.

[0090] Grasp force test: The grip force was measured using a grip force measuring instrument. The mouse's torso was kept parallel to the grid, and its hind limbs were allowed to grasp the measuring instrument. The mouse's tail was then grasped and pulled backward evenly, and the maximum grip force value was recorded. This was repeated 10 times for each mouse, and the average value was taken as the final grip force value for that mouse.

[0091] The results showed that mice injected with PLE4-OSKM lentivirus suspension had significantly increased grip strength compared to mice injected with PLE4-GAL4 lentivirus suspension. Figure 1 The results (C) indicate that the PLE4-OSKM lentivirus suspension enhances the motor function of mice.

[0092] 3.1.2 Detection of OSKM (Oct4, Sox2, Klf4, and C-Myc) expression levels in cartilage tissue

[0093] Right posterior knee cartilage tissue from PLE4-GAL4 and PLE4-OSKM mice was lysed using TRIzol (Invitrogen, 15596018) and RNA was extracted. RT-qPCR was used to detect the transcriptional (mRNA) levels of Oct4, Sox2, Klf4, and C-Myc. Primer sequences are as follows:

[0094] Oct4-F: 5'-GGCTTCAGACTTCGCCTTCT-3';

[0095] Oct4-R: 5'-TGGAAGCTTAGCCAGGTTCG-3';

[0096] Sox2-F: 5'-TTTGTCCGAGACCGAGAAGC-3';

[0097] Sox2-R: 5'-CTCCGGGAAGCGTGTACTTA-3';

[0098] Klf4-F: 5'-GCACACCTGCGAACTCACAC-3';

[0099] Klf4-R: 5'-CCGTCCCAGTCACAGTGGTAA-3';

[0100] C-Myc-F: 5'-ACCACCAGCAGCGACTCTGA-3';

[0101] C-Myc-R: 5'-TGCCTCTTCTCCACAGACACC-3';

[0102] Compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension showed significantly increased mRNA levels of Oct4, Sox2, Klf4, and C-Myc in cartilage tissue. Figure 1 (B) indicates that the injection of PLE4-OSKM lentiviral suspension into the joint cavity successfully infected chondrocytes and increased the expression levels of Oct4, Sox2, Klf4 and C-Myc.

[0103] 4. Effect Evaluation

[0104] Eight weeks after injecting a suspension of PLE4-GAL4 and PLE4-OSKM lentivirus into the left hind limb knee joint of mice, muscle tissue was removed, leaving a 1 cm length of femur and tibia. The bone was then fixed in 4% paraformaldehyde solution for CT detection and tissue embedding staining. The specific steps are as follows:

[0105] 4.1 CT scan

[0106] Knee joints fixed in 4% paraformaldehyde were scanned using a small animal CT scanner (Quantum GX2) at a voltage of 52 kVp, a current of 100 μA, and a resolution of 36 μm. Osteophyte volume was analyzed using Analyze (V14.0) and Slicer (V5.2) software.

[0107] CT scan data showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly reduced bone volume (BV) at the joints. Figure 1 (D). This indicates that supplementing the joint cavity with OSKM can help inhibit bone hyperplasia.

[0108] 4.2 Safranin and Fast Green Staining of Articular Cartilage

[0109] (1) After dewaxing and rehydrating the sections, immerse them in fresh hematoxylin staining solution at room temperature for 3-5 minutes.

[0110] (2) After differentiation with 1% hydrochloric acid / ethanol for 15 seconds, rinse with distilled water for 10 minutes.

[0111] (3) Place it in the Fast Green staining solution and immerse it at room temperature for 5 minutes.

[0112] (4) Quickly wash with a weak acid solution for 10-15 seconds to remove residual solid green, and then air dry.

[0113] (5) Place in safranin staining solution and immerse at room temperature for 5 minutes.

[0114] (6) Dehydration, clearing, and sealing.

[0115] (7) Scan and count.

[0116] Safranin-Fix Green staining results showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had a significantly increased cartilage area. Figure 1 (E). This indicates that increasing the content of OSKM in cartilage tissue helps promote cartilage tissue growth.

[0117] 4.3 Hematoxylin-eosin (H&E) staining of articular cartilage tissue

[0118] (1) After dewaxing and rehydrating the sections, immerse them in fresh hematoxylin staining solution at room temperature for 3-5 minutes.

[0119] (2) After differentiation with 1% hydrochloric acid / ethanol for 15 seconds, rinse with distilled water for 10 minutes.

[0120] (3) Place it in eosin staining solution and immerse it at room temperature for 5 minutes.

[0121] (4) Rinse with clean water for 2 minutes.

[0122] (5) Dehydration, clearing, and sealing.

[0123] (6) Scan and count.

[0124] H&E staining results showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly reduced synovial thickness. Figure 1 (F). This indicates that supplementing the joint cavity with OSKM helps inhibit synovial hyperplasia.

[0125] 4.4 Immunohistochemical staining of articular cartilage tissue

[0126] (1) Take the fixed joint tissue and decalcify it at 4°C using a decalcification solution containing 5% formic acid until the femur and tibia tissue softens and can be punctured with a needle (about 7-10 days).

[0127] (2) Embedded in paraffin, sectioned, with a thickness of 5μm, and baked at 60℃ overnight.

[0128] (3) Dewax and rehydrate the sections.

[0129] (4) Use 0.125% trypsin (ZLI-9010) to repair the antigen and incubate at 37°C for 30 minutes.

[0130] (5) Use 0.4% Triton X-100 (Sigma-Aldrich, T9284) to allow it to pass through at room temperature for 1 hour.

[0131] (6) Use endogenous peroxidase blockers to block endogenous peroxidase.

[0132] (7) After washing with PBS at room temperature, add primary antibody and incubate overnight at 4°C.

[0133] (8) Wash with PBS at room temperature, 10 minutes each time, for 3 times.

[0134] (9) Add reaction enhancement solution dropwise and incubate at room temperature for 20 minutes.

[0135] (10) Wash thoroughly with PBS at room temperature, 20 minutes each time, for a total of 3 times.

[0136] (11) Add enzyme-labeled goat anti-mouse / rabbit IgG polymer (Zhongshan Jinqiao Company, PV-9001 / PV-9002) and incubate at room temperature for 1 hour.

[0137] (12) Select an appropriate time for DAB color development.

[0138] (13) Hematoxylin staining, differentiation, rinsing and blue staining.

[0139] (14) Dehydration, clearing, and sealing.

[0140] (15) The images were scanned using a PE panoramic scanner (PerkinElmer Vectra Polaris) and the proportion of positive cells in the cartilage layer was statistically analyzed using Image J.

[0141] The antibody used to detect the P21 protein was manufactured by Cell Signaling Technology, catalog number 2947S.

[0142] The antibody used to detect MMTV protein was manufactured by Novus Biologicals, catalog number NBP2-44179.

[0143] The antibody used to detect IL-6 protein was manufactured by Abcam, catalog number ab6672.

[0144] The antibody used to detect TNFα protein was manufactured by Abcam, catalog number ab1793.

[0145] The antibody used to detect MMP13 protein was manufactured by Abcam, catalog number ab39012.

[0146] The antibody used to detect Aggrecan protein was manufactured by ABclonal, catalog number A11691.

[0147] The results showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly reduced proportions of P21 and MMTV positive cells. Figure 1 In G and H), IL-6 and TNFα-positive cells were significantly reduced ( Figure 1 The results (I and J) indicate that supplementing the joint cavity with OSKM significantly reduced senescent and inflammatory cells in cartilage tissue. Furthermore, the proportion of positive cells secreting the proteoglycan Aggrecan significantly increased. Figure 1 The proportion of positive cells secreting matrix metalloproteinase MMP13 was significantly reduced (K), while the proportion of positive cells was significantly reduced (K). Figure 1 The result (L) indicates that supplementing the joint cavity with OSKM significantly increases the number of cells in the cartilage tissue that secrete the extracellular matrix necessary for cartilage.

[0148] Example 1 illustrates that supplementing OSKM alleviates articular cartilage aging in aged mice, reduces cartilage inflammation levels, and enhances the bioactivity of cartilage tissue cells.

[0149] Example 2: The therapeutic effect of increasing OSKM content on pathological osteoarthritis in mice.

[0150] 1. Preparation of lentivirus expressing mouse OSKM

[0151] Same as step 1 in Example 1.

[0152] 2. Establish an anterior cruciate ligament injury-induced osteoarthritis (ACLT) model.

[0153] 2.1 Establishment of ACLT mouse model

[0154] Test mice: Female, 2-month-old SPF-grade mice (C57BL / 6J), from Spifort (Beijing) Biotechnology Co., Ltd. Mice housing conditions: 23℃, 12-hour light / 12-hour dark, free access to food and water. Experimental equipment and materials included: isoflurane gas anesthesia device, insulin injector.

[0155] Mice were randomly divided into 3 groups, with 12 mice in each group. Two groups of mice were randomly selected for ACLT modeling:

[0156] After anesthetizing the mice, the skin was cut open at the knee joint of the hind limb to expose the knee joint, the anterior cruciate ligament was cut, the skin was sutured, and the ACLT mouse model was obtained after 10 days of normal feeding.

[0157] The other group received no treatment and served as the control group (Sham group).

[0158] 2.2 Lentiviral injection

[0159] Sham group: No virus injection.

[0160] PLE4-GAL4 group: 20 μl of PLE4-GAL4 lentivirus suspension was injected into the left and right joint cavities of each mouse's hind limb.

[0161] PLE4-OSKM group: 20 μl of PLE4-OSKM lentivirus suspension was injected into the joint cavity of each mouse's hind limb.

[0162] 3. Effectiveness Evaluation

[0163] 8 weeks after injection of lentivirus suspension ( Figure 2 (A) The efficacy of the treatment was evaluated. The specific method was as follows: Mice were euthanized by spinal dislocation, and the hind limb knee joint was isolated. After removing muscle tissue from the left knee joint, a 1cm length of femur and tibia was retained for CT examination and tissue embedding staining. The changes in joint tissue pathology were evaluated according to step 4 of Example 1, where the OARSI cartilage scoring criteria were as follows:

[0164] 0 = Normal;

[0165] 0.5 = No structural change, a small portion of the green (proteoglycan) was lost;

[0166] 1 = Slight wear on the cartilage surface, with the damaged area less than 5% of the total area;

[0167] 2 = Partial loss of cartilage surface matrix, not involving deep cartilage, with the damaged area accounting for about 5%-10% of the total area;

[0168] 3. The damage involves the calcified cartilage layer, and the damaged area accounts for about 10%-24% of the total area;

[0169] 4. Damage extends below the calcified cartilage, resulting in the loss of 25%-50% of the cartilage beyond the joint surface;

[0170] 5 = 50%-75% loss of cartilage on the joint surface;

[0171] 6 = More than 75% of the cartilage on the joint surface is lost, and only a small amount of acellular collagen area may remain.

[0172] CT scan data showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly reduced joint osteophyte volume. Figure 2 (B) This indicates that supplementing the joint cavity with OSKM can help inhibit bone hyperplasia.

[0173] Safranin-Fix Green staining results showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had lower cartilage OARSI scores. Figure 2 (C) This indicates that increasing the content of OSKM in cartilage tissue helps promote cartilage tissue growth.

[0174] H&E staining results showed that, compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly reduced synovial thickness. Figure 2 (D). This indicates that supplementing the joint cavity with OSKM helps inhibit synovial hyperplasia.

[0175] Simultaneously, it was found that the proportion of cells secreting the polysaccharide Aggrecan was significantly increased. Figure 2 In the middle E), the proportion of cells secreting matrix metalloproteinase MMP13 was significantly reduced ( Figure 2 The presence of IL-6 positive cells (F) indicates that supplementing the joint cavity with OSKM increases the number of cells secreting the extracellular matrix necessary for cartilage in the cartilage tissue. Furthermore, the number of IL-6 positive cells significantly decreased. Figure 2 The presence of OSKM in the joint cavity (G) indicates that supplementing the joint cavity with OSKM reduces inflammatory cells in the cartilage tissue.

[0176] Example 2 illustrates that supplementing the joint cavity with OSKM alleviates cartilage wear caused by pathological osteoarthritis, reduces joint inflammation levels, and improves the bioactivity of cartilage tissue cells.

[0177] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances that increase the content or activity of OSKM in the preparation of products for treating osteoarthritis; The substance that increases the content or activity of OSKM is a biomaterial related to OSKM; The biological material is PLE4-OSKM, which is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the OSKM gene shown in SEQ ID No. 1 of the sequence listing. The osteoarthritis mentioned is age-related osteoarthritis.

2. The application according to claim 1, characterized in that: The treatment for osteoarthritis focuses on reducing the level of inflammation in the cartilage.

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