A method for genetically engineering anti-aging human mesenchymal stem cells and application thereof

Abstract

The application belongs to the technical field of genetic engineering and cell modification, and discloses a genetic engineering construction method and application of an anti-aging human mesenchymal stem cell. Based on eliminating R-loop formed in the process of cell aging from the source, relieving the adverse effects of genome instability caused by R-loop, the application develops RNH1 overexpression virus transfection MSC aiming at eliminating cell R-loop. The RNH1 overexpression MSC provided by the application exhibits significant anti-aging activity, has strong environmental adaptability and excellent safety characteristics, can effectively resist harsh in-vivo inflammation and aging microenvironment, and avoid the tumorigenic risk after cell transplantation, and the MSCs with stable anti-aging ability can be applied to significantly improve the curative effect of MSCs infusion in-vivo disease treatment.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and cell modification technology, and to a method for constructing mesenchymal stem cells, specifically to a genetic engineering method for constructing and applying anti-aging human mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent adult stem cells derived from the mesoderm, found in tissues such as bone marrow, adipose tissue, umbilical cord, placenta, dental pulp, and synovium. They can expand in vitro for extended periods, differentiating into various cell types including bone, cartilage, adipose tissue, nerve cells, cardiomyocytes, and hepatocytes. They do not express HLA-DR antigen (human leukocyte antigen-DR), but only weakly express HLA-A / B (human leukocyte antigen-B). Allogeneic transplantation does not require strict matching and rarely triggers immune rejection. They can regulate the microenvironment and promote tissue regeneration through inflammatory and homing effects, as well as secretory effects. Mesenchymal stem cell infusion therapy has shown significant potential in the treatment of various diseases and has been used for autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis; musculoskeletal diseases such as osteoarthritis; endocrine system diseases such as type II diabetes; circulatory system diseases such as acute myocardial infarction and heart failure; and neurological diseases such as spinal cord injury and brain injury. Senescence in mesenchymal stem cells (MSCs) refers to the biological process by which, under physiological or pathological conditions, cumulative damage leads to a gradual decline in their self-renewal capacity, multi-lineage differentiation potential, and paracrine function. This process is accompanied by characteristic changes in cell morphology, gene expression, and metabolic activity, ultimately manifesting as a loss of repair and regeneration capabilities. It is one of the important mechanisms of overall aging and the development of age-related diseases.

[0003] Although significant progress has been made in the study of mesenchymal stem cell (MSC) senescence in recent years, the mechanisms of cellular senescence remain highly complex. The exact causes of cellular senescence are still unclear, and targeted interventions for MSC senescence remain elusive. For example, while current antioxidants (such as vitamin C) or autophagy activators (such as rapamycin) can reverse MSC senescence to some extent in vitro, the in vivo microenvironment is complex, containing factors such as oxidative stress and chronic inflammation. Cells entering the in vivo microenvironment may be altered by these factors, leading to a significant reduction in efficacy. Furthermore, in vitro culture conditions cannot simulate the dynamic changes in the in vivo aging microenvironment, such as inflammatory factor gradients and intercellular communication interference. Simultaneously, drug-based in vitro cell interventions have a limited time-sensitivity and cannot achieve stable anti-aging effects. Current genetic engineering often targets single gene loci for overexpression or knockout, but the complex multi-gene interaction networks during cellular senescence make it difficult to alter overall cellular function through single-gene editing.

[0004] Therefore, this invention provides a genetic engineering method for constructing and applying anti-aging human mesenchymal stem cells. Summary of the Invention

[0005] This invention aims to provide a genetic engineering construction method and application of anti-aging human mesenchymal stem cells. By overexpressing RNH1, stable anti-aging MSCs are constructed, solving the technical problem in the prior art that it is difficult to construct stable and effective MSCs with anti-aging ability after in vivo infusion.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides a method for constructing genetically engineered human mesenchymal stem cells overexpressing RNH1, comprising the following steps:

[0008] S1. Insert the target gene RNH1 into a lentiviral system vector to construct the GV492-RNH1 recombinant plasmid. The amino acid sequence of the target gene RNH1 is shown in SEQ ID NO.1.

[0009] S2. The GV492-RNH1 recombinant plasmid obtained in S1 and the packaging plasmid were co-transfected into host cells to prepare lentiviral particles.

[0010] S3. Transfect human mesenchymal stem cells with the lentiviral particles obtained in S2 to obtain genetically engineered human mesenchymal stem cells that overexpress RNH1 for anti-aging or anti-inflammatory purposes.

[0011] Preferably, the construction method of the GV492-RNH1 recombinant plasmid is as follows: after PCR amplification using primer pairs, the target gene RNH1 is cloned; then the lentiviral system vector and the RNH1 gene sequence are digested with AgeI and NheI restriction endonucleases, respectively, and the cloning ligation is completed by the in-fusion seamless cloning method.

[0012] Preferably, the primer pair includes a forward primer: 5'-ATGAAAGCGCAGAGCCGTAT-3' and a reverse primer: 5'-CTCCCATGTAGGAAAACGTGTC-3'.

[0013] Preferably, the lentiviral system vector is GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin).

[0014] Preferably, the host cell is a 293T cell, and the packaging plasmids are psPAX2 and pMD2.G.

[0015] Preferably, in step S3, the transfection specifically involves: in a solution containing 1 x 10 5Add a mixture of RNH1 overexpression lentivirus, transfection aid Polybrene, and DMEM complete medium in a ratio of 10:86:4 to each well of MSCs and incubate for 48 hours.

[0016] On the one hand, the present invention provides genetically engineered human mesenchymal stem cells overexpressing RNH1 prepared by the above-described construction method.

[0017] On the one hand, the present invention provides the application of the above-mentioned human mesenchymal stem cells overexpressing RNH1 in the preparation of anti-aging and anti-inflammatory drugs.

[0018] Preferably, the drug refers to a drug that achieves anti-aging or anti-inflammatory effects by upregulating RNH1 levels, downregulating R-loop levels, downregulating SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6 and TGF-β levels, upregulating ATP levels and downregulating OCR levels.

[0019] Preferably, the drug comprises overexpressing RNH1 or silencing the R-loop.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention addresses the issue of eliminating R-loops (R-loops) formed during cellular senescence at their source, mitigating the adverse effects of genomic instability caused by R-loops. It involves developing an RNH1-overexpressing virus transfected with MSCs to eliminate cellular R-loops. This is the first time a strategy has been proposed to stabilize RNH1 overexpression and thereby eliminate genomic R-loop structures, enhancing the anti-aging ability of MSCs after in vivo infusion. These MSCs exhibit stable RNH1 overexpression and anti-aging functions, and their approach is not targeted at a single gene locus but rather alleviates a range of aging phenotypes and genomic instability issues by eliminating R-loops. RNH1-overexpressing MSCs demonstrate significant anti-aging activity, strong environmental adaptability, and excellent safety profiles. They effectively resist harsh in vivo inflammation and the aging microenvironment while avoiding the risk of tumorigenesis after cell transplantation. These MSCs with stable anti-aging capabilities can subsequently be used to significantly improve the therapeutic efficacy of MSCs in vivo for disease treatment. Attached Figure Description

[0022] Figure 1 This is a staining map and quantitative analysis of β-galactosidase staining to detect the aging level of MSCs in different groups (young refers to young primary MSCs, and old refers to old primary MSCs, UV). - This refers to young, passaged third-generation MSCs that have not undergone UV-induced aging. +P3 refers to young passaged third-generation MSCs that have undergone UV-induced aging, while P12 refers to naturally aging young passaged 12 MSCs.

[0023] Figure 2 It is an immunoblotting and quantitative analysis of P16 and P21 protein expression in MSCs from different groups using Western blotting.

[0024] Figure 3 These are the results of RT-qPCR detection of the expression levels of P16 and P21 mRNA in MSCs from different groups;

[0025] Figure 4 The results are from RT-qPCR detection of the expression levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6 and TGF-β mRNA in different groups of MSCs;

[0026] Figure 5 The results are obtained by ELISA detection of the secretion levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6 and TGF-β proteins in different groups of MSCs;

[0027] Figure 6 These are the results of ATP level detection in different groups of MSCs;

[0028] Figure 7 These are the results of OCR (oxygen consumption rate) level tests for different groups of MSCs;

[0029] Figure 8 This is a fluorescence image and quantitative analysis of the R-loop level of MSCs in different groups detected by S9.6 immunofluorescence.

[0030] Figure 9 It is an immunoblot chromatogram and quantitative analysis of R-loop levels of MSCs in different groups using S9.6 Dot blot;

[0031] Figure 10 The results are from transcriptome sequencing of RNH1 expression levels in MSCs of different groups;

[0032] Figure 11 This is a flowchart of a mouse model of CIA arthritis treated with OE-RNH1-MSCs (RNH1-overexpressing MSCs) infusion;

[0033] Figure 12 These are the results of Western blotting and quantitative analysis of RNH1 protein immunoblotting in different groups of MSCs (Vector refers to empty vector, and OE-RNH1 refers to viral vector overexpressing RNH1).

[0034] Figure 13 This is an S9.6 immunofluorescence assay showing the fluorescence pattern and quantitative analysis of R-loop levels in R-loop MSCs overexpressing RNH1 in different groups (aged refers to aged primary MSCs, aged +OE-RNH1 refers to aged primary MSCs overexpressing RNH1, UV...). + P12 refers to naturally aged MSCs passaged to the 12th generation after UV-induced aging; P12+OE-RNH1 refers to naturally aged MSCs passaged to the 12th generation after UV-induced aging and overexpressing RNH1.

[0035] Figure 14 This is an immunoblot and quantitative analysis of the R-loop levels of RNH1-overexpressing MSCs in different groups using S9.6 Dot blot.

[0036] Figure 15 This is a staining pattern and quantitative analysis of β-galactosidase staining to detect the senescence level of RNH1-overexpressing MSCs in different groups;

[0037] Figure 16 The results are obtained by RT-qPCR detection of the expression levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6 and TGF-β in different groups of RNH1 MSCs overexpressing RNH1.

[0038] Figure 17 The results are obtained by ELISA detection of the secretion levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6 and TGF-β proteins in different groups of RNH1-overexpressing MSCs;

[0039] Figure 18 These are the ATP levels detected in MSCs overexpressing RNH1 in different groups;

[0040] Figure 19 These are the results of the detection of OCR (oxygen consumption rate) levels in RNH1-overexpressing MSCs from different groups;

[0041] Figure 20 These are gross images of the ankle joint of mice after OE-RNH1-MSCs treatment, along with tissue HE staining and Safranin-Fix-Green staining results.

[0042] Figure 21 These are the results of arthritis scores and palm thickness measurements in mice after OE-RNH1-MSCs treatment. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] Example 1: Extraction of MSCs to construct a senescent cell model

[0047] 1. Experimental Methods

[0048] 1) Construction of senescent cell model

[0049] Construction of a primary senescent cell model: 30 ml of bone marrow was extracted from elderly and young healthy volunteers via the posterior superior iliac spine, and anticoagulated with heparin. The bone marrow was mixed with PBS and centrifuged (1500 rpm, 5 min) to remove the supernatant and lipids. The cell suspension was slowly added to the upper layer of the lymphocyte separation medium and centrifuged (2000 rpm, 20 min); the white membrane layer containing MSCs was aspirated. The cells were washed twice with PBS, resuspended, and seeded in medium containing 15% FBS for culture. MSCs were passaged to the third generation at a rate of 1 × 10⁻⁶ cells / mL. 5 The MSCs were seeded at a density of / wells in 6-well plates to obtain aged and young passaged MSCs.

[0050] Construction of a passaged senescent cell model: Young MSCs were passaged to the 12th generation, at a rate of 1×10⁻⁶ cells / cells. 5 / well density seeding in 6-well plates to obtain young passaged naturally senescent MSCs.

[0051] Construction of a UV-induced senescent cell model: Young primary MSCs were passaged to the 3rd generation, at a ratio of 1×10⁻⁶ cells / cells. 5 MSCs were seeded at a density of / wells in 6-well plates. After a single irradiation with UVB (medium-wave ultraviolet light, 280–320 nm) at 40 mJ / cm², MSCs were cultured for 72 hours to obtain UV-induced senescent MSCs.

[0052] 2) Measure various indicators within the senescent cell models MSCs constructed in 1).

[0053] The senescence level of MSCs was detected using a β-galactosidase staining kit, the expression of P16 / P21 protein in MSCs was detected using Western blot, and the expression of P16 / P21 mRNA in MSCs was detected using RT-qPCR.

[0054] SASP mRNA expression in MSCs was detected using RT-qPCR, and SASP secretion in MSCs was detected using ELISA kits. ATP levels in MSCs were detected using ATP assay kits, and OCR (oxygen consumption rate) levels in MSCs were detected using OCR assay kits. Intracellular R-loop levels in MSCs were detected using immunofluorescence staining and Dot blot.

[0055] 2. Experimental Results

[0056] 2.1 Senescence levels in aged and young MSCs stained with β-galactosidase

[0057] β-galactosidase staining results showed that, compared to the younger group of MSCs, the older group of MSCs had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells; compared to the UV- group of MSCs, the UV+ group of MSCs had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells; compared to the P3 group of MSCs, the P12 group of MSCs had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells (see...). Figure 1 ).

[0058] 2.2 Western Blot and RT-qPCR detection of P16 / P21 expression in aged and young MSCs

[0059] Western blot and RT-qPCR results showed that, compared with the younger group of MSCs, the older group of MSCs had higher expression levels of P16 and P21 proteins and mRNA; compared with the UV- group of MSCs, the UV+ group of MSCs had higher expression levels of P16 and P21 proteins and mRNA; compared with the P3 group of MSCs, the P12 group of MSCs had higher expression levels of P16 and P21 proteins and mRNA (see...). Figure 2 and Figure 3 ).

[0060] 2.3 RT-qPCR and LISA detection of SASP expression and secretion in aged and young MSCs

[0061] Compared to younger MSCs, older MSCs showed higher expression levels and protein secretion levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA. Compared to the UV- group MSCs, the UV+ group MSCs showed higher expression levels and protein secretion levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA. Compared to the P3 group MSCs, the P12 group MSCs showed higher expression levels and protein secretion levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA. Figure 4 and Figure 5 ).

[0062] 2.4 ATP Level Detection: ATP levels in aged and young MSCs

[0063] Compared to younger MSCs, older MSCs had lower ATP levels; compared to UV- group MSCs, UV+ group MSCs had lower ATP levels; compared to P3 group MSCs, P12 group MSCs had lower ATP levels (see...). Figure 6 ).

[0064] 2.5 OCR Level Detection of Oxidative Respiration Levels in Aged and Young MSCs

[0065] Compared to younger MSCs, older MSCs had lower OCR levels; compared to UV- MSCs, UV+ MSCs had lower OCR levels; compared to P3 MSCs, P12 MSCs had lower OCR levels (see...). Figure 7 ).

[0066] 2.6 S9.6 immunofluorescence and S9.6 dot blot detection of R-loop levels in aged and young MSCs

[0067] The results all showed that, compared to the younger group, the older group had higher R-loop levels in MSCs; compared to the UV- group, the UV+ group had higher R-loop levels in MSCs; and compared to the P3 group, the P12 group had higher R-loop levels in MSCs (see...). Figure 8 and Figure 9 ).

[0068] In summary, senescent MSCs exhibit elevated R-loop levels, high β-galactosidase staining quantification, high levels of P16 / P21 protein and mRNA, high SASP expression and secretion levels, and lower ATP and OCR levels.

[0069] Example 2: Construction and application of RNH1 overexpression lentivirus

[0070] This embodiment, through transcriptome sequencing data, found that the TPM value of RNH1 was significantly lower in both young and old MSCs, indicating that the RNA helicase protein RNH1 is significantly downregulated in elderly mesenchymal stem cells. Figure 10 In other words, RNH1 downregulation mediates the accumulation of genomic R-loop. Therefore, an RNH1-overexpressing lentivirus was constructed to verify the effect of RNH1 overexpression on the anti-aging effect of mesenchymal stem cells. RNH1-overexpressing MSCs with stable anti-aging ability were used for infusion therapy in CIA arthritis model mice and achieved good therapeutic results.

[0071] I. Construction of RNH1 overexpression lentivirus

[0072] (1) Plasmid construction:

[0073] The human RNH1 gene was obtained from a cDNA library of Genechem (Shanghai Genechem Medical Technology Co., Ltd.) using the following primers: RNH1 forward primer: 5'-ATGAAAGCGCAGAGCCGTAT-3', RNH1 reverse primer: 5'-CTCCCATGTAGGAAAACGTGTC-3'; the RNH1 gene with the cloned amino acid sequence is shown in SEQ ID NO.1.

[0074] The lentiviral vector plasmid GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin) (purchased from Shanghai Genomics Co., Ltd.) and the RNH1 gene sequence were digested with AgeI and NheI restriction endonucleases, respectively, and cloning was completed using the in-fusion seamless cloning (recombination) method. The recombinant vector was identified by DNA sequencing.

[0075] The amino acid sequence of RNH1 (SEQ ID NO.1) is as follows:

[0076] MSWLLLFLAHRVALAALPCRRGSRGFGMFYAVRRGRKTGVFLTWNECRAQVDRFPAARFKKFATEDEAWAFVRKSASPEVSEGHENQHGQESEAKASKRLREPLDGDGHESAEPYAKHMKPSVEPAPPVSRDTFSYMGDFVVVY TDGCCSNGRRRPRAGIGVYWGPGHPLNVGIRLPGRQTNQRAEHAACKAIEQAKTQNINKLVLYTDSMFTINGITNWVQGWKKNGWKTSAGKEVINKEDFVALERLTQGMDIQWMHVPGHSGFIGNEEADRLAREGAKQSED.

[0077] (2) Lentiviral production:

[0078] The constructed viral vector, along with two helper packaging plasmids, psPAX2 and pMD2.G, was co-transfected into 293T cells using Lipofectamine 2000 (purchased from Invitrogen; Thermo Fisher Scientific, Inc.). 72 hours after transfection, the virus-containing culture supernatant was collected, rapidly centrifuged at 1000×g to remove cell debris, and then filtered through a 0.45 μm cellulose acetate membrane for sterilization.

[0079] Viral titer was determined by flow cytometry (FACS) analysis of the proportion of GFP-positive 293T cells, and was approximately 1 × 10⁻⁶. 9 Transduction units (TU) / mL of culture medium.

[0080] II. Application of RNH1 overexpression lentiviruses

[0081] 1. Experimental Methods

[0082] (1) In vitro cell experiments

[0083] 1) Young and aged primary MSCs, passaged induced senescent MSCs, and UV-induced senescent MSCs prepared in Example 1 were transfected with RNH1-overexpressing lentivirus for 48 hours, respectively. Specifically, 1 x 10 5 After seeding MSCs into 12-well plates, aspirate the supernatant, mix RNH1 overexpression lentivirus, transfection aids, and complete culture medium in a ratio of 10:86:4, add 2 ml / well, and incubate for 48 hours.

[0084] 2) Western blot was used to detect RNH1 protein expression in MSCs, and RT-qPCR was used to detect RNH1 mRNA expression in MSCs. Immunofluorescence staining and Dot blot were used to detect intracellular R-loop levels in MSCs. β-galactosidase staining was used to detect MSC senescence levels. Western blot was used to detect P16 / P21 protein expression in MSCs. RT-qPCR was used to detect P16 / P21 mRNA expression in MSCs. RT-qPCR was used to detect SASP mRNA expression in MSCs, and ELISA was used to detect SASP secretion in MSCs. ATP levels in MSCs were detected using an ATP assay kit. OCR levels in MSCs were detected using an OCR assay kit.

[0085] (2) In vivo animal experiments

[0086] 1) Construct a mouse model of CIA arthritis. The specific construction plan is as follows:

[0087] On day 1, male DBA / 1 mice (6-8 weeks old, weighing 20-30 g) were subcutaneously injected with a 100 μL homogenized emulsion mixture prepared from chicken type II collagen (Chondrex, #20012) and an equal volume of Freund's complete adjuvant (Chondrex, #7001). A booster immunization was performed on day 21 using a 100 μL emulsion mixture prepared from chicken type II collagen and Freund's incomplete adjuvant (Chondrex, #7002). The injection was administered slowly at the base of the tail, carefully avoiding areas with large, visible blood vessels at the initial injection site.

[0088] Arthritis severity assessment:

[0089] Arthritis scoring for each leg (limb) of the mouse was performed independently, with the following scoring criteria: 0 points: normal; 1 point: redness or swelling of a single joint; 2 points: redness or swelling of multiple joints; 3 points: redness or swelling of the entire foot; 4 points: joint stiffness or severe deformity. The total arthritis score for each mouse was the sum of the scores for all four legs.

[0090] Hind paw thickness measurement: The thickness of the mouse's hind paw was measured using vernier calipers to assess the severity of inflammation.

[0091] 2) OE-RNH1-MSCs infusion therapy in a mouse model of CIA arthritis

[0092] For detailed procedures, please see [link / document / documentation]. Figure 11After successful construction, the system was used to detect local gross images of mouse ankle joints, perform HE staining and safranin-fast green staining, detect arthritis scores, and measure palm thickness.

[0093] 2. Experimental Results

[0094] (1) In vitro cell experiments

[0095] Western blot analysis showed that RNH1 protein levels were overexpressed in the OE-RNH1 group: compared to the Vector group MSCs, the OE-RNH1 group MSCs showed significantly increased RNH1 protein expression through immunoblotting and quantification (see [link to data]). Figure 12 ).

[0096] S9.6 Immunofluorescence and Dot blot analysis showed a significant decrease in R-loop levels in RNH1-overexpressing MSCs: The R-loop levels were lower in the elderly + OE-RNH1 group compared to the elderly group; lower in the UV + OE-RNH1 group compared to the UV + group; and lower in the P12 + OE-RNH1 group compared to the P12 group (see [link to data]). Figure 13 and 14 ).

[0097] β-galactosidase staining showed a significant decrease in senescence levels in RNH1-overexpressing MSCs: compared to the aged group MSCs, the aged +OE-RNH1 group MSCs stained lighter and had a lower proportion of positive cells; compared to the UV+ group MSCs, the UV+OE-RNH1 group MSCs stained lighter and had a lower proportion of positive cells; compared to the P12 group MSCs, the P12+OE-RNH1 group MSCs stained lighter and had a lower proportion of positive cells (see...). Figure 15 ).

[0098] RT-qPCR and ELISA showed significantly reduced SASP expression and secretion levels in RNH1-overexpressing MSCs: Compared to the elderly group MSCs, the elderly + OE-RNH1 group MSCs had lower levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA expression and protein secretion; compared to the UV+ group MSCs, the UV+OE-RNH1 group MSCs had lower levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA expression and protein secretion; compared to the P12 group MSCs, the P12+OE-RNH1 group MSCs had lower levels of SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β mRNA expression and protein secretion (see [link to study]). Figure 16 and 17).

[0099] ATP assays showed significantly elevated ATP levels in MSCs overexpressing RNH1: MSCs in the aged + OE-RNH1 group had higher ATP levels than those in the aged group; MSCs in the UV + OE-RNH1 group had higher ATP levels than those in the UV + group; and MSCs in the P12 + OE-RNH1 group had higher ATP levels than those in the P12 group (see [link to study].) Figure 18 ).

[0100] OCR detection showed significantly elevated OCR levels in RNH1-overexpressing MSCs: The OCR levels were higher in the aged + OE-RNH1 group compared to the aged group; higher in the UV + OE-RNH1 group compared to the UV + group; and higher in the P12 + OE-RNH1 group compared to the P12 group (see [link to OCR analysis]). Figure 19 ).

[0101] In summary, overexpression of RNH1 significantly upregulated RNH1 mRNA and protein levels, significantly downregulated R-loop levels, decreased β-galactosidase staining quantification, weakened P16 / P21 protein and mRNA levels, weakened SASP expression and secretion levels, and increased ATP and OCR levels in MSCs, demonstrating that overexpression of RNH1 can significantly promote the aging resistance of MSCs.

[0102] (2) In vivo animal experiments

[0103] Gross images of the ankle joint, HE staining, and Safranin-Fix-Green staining of a mouse model of CIA arthritis treated with OE-RNH1-MSCs infusion: Compared with the CIA group and the CIA+MSCs infusion treatment group, the CIA+OE-RNH1-MSCs infusion treatment group showed less ankle swelling, less synovial hyperplasia, less inflammatory cell infiltration, and less joint destruction (see...). Figure 20 ).

[0104] OE-RNH1-MSCs infusion treatment of a mouse model of CIA arthritis: Arthritis score and palm thickness measurement: Compared with the CIA group and the CIA+MSCs infusion treatment group, the CIA+OE-RNH1-MSCs infusion treatment group had lower arthritis scores and thinner palms (see...). Figure 21 ).

[0105] In summary, OE-RNH1-MSCs infusion therapy in a CIA mouse model of arthritis resulted in relief of local joint inflammation and reduction in arthritis scores and palm thickness.

[0106] In summary, this invention utilizes a strategy of stably overexpressing RNH1 via lentivirus to target the elimination of genomic R-loop structures in the development of anti-aging human mesenchymal stem cells (MSCs). This strategy does not target a specific gene, but rather focuses on the core mechanism mediating cellular senescence—the exacerbation of genomic instability. By overexpressing RNH1 to eliminate the key culprit behind this instability—the R-loop structure—anti-aging MSCs are constructed. Stable RNH1-overexpressing MSCs, by stably eliminating intracellular R-loop structures, are expected to better cope with various stimuli inducing cellular senescence in the complex in vivo microenvironment after infusion, thereby stably exerting their therapeutic effect and improving in vivo MSC infusion therapy.

[0107] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Application of anti-aging human mesenchymal stem cells in the preparation of anti-arthritis drugs, wherein the anti-aging human mesenchymal stem cells are derived from bone marrow and are human mesenchymal stem cells overexpressing RNH1; the amino acid sequence of RNH1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The method for constructing anti-aging human mesenchymal stem cells includes the following steps: S1. Insert the target gene RNH1 into the lentiviral system vector to construct the GV492-RNH1 recombinant plasmid. S2. The GV492-RNH1 recombinant plasmid obtained in S1 and the packaging plasmid were co-transfected into host cells to prepare lentiviral particles. S3. Transfect human mesenchymal stem cells with the lentiviral particles obtained in S2 to obtain anti-aging human mesenchymal stem cells.

3. The application according to claim 2, characterized in that, The construction method of the GV492-RNH1 recombinant plasmid is as follows: after PCR amplification using primer pairs, the target gene RNH1 is cloned; then the lentiviral system vector and the RNH1 gene sequence are digested with AgeI and NheI restriction endonucleases, respectively, and the cloning ligation is completed by the in-fusion seamless cloning method.

4. The application according to claim 3, characterized in that, The primer pair includes a forward primer: 5'-ATGAAAGCGCAGAGCCGTAT-3' and a reverse primer: 5'-CTCCCATGTAGGAAAACGTGTC-3'.

5. The application according to claim 3, characterized in that, The lentiviral system vector is pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.

6. The application according to claim 2, characterized in that, The host cell was a 293T cell.

7. The application according to claim 2, characterized in that, The packaging plasmids are psPAX2 and pMD2.G.

8. The application according to claim 2, characterized in that, In step S3, the transfection specifically involves: in a solution containing 1 x 10 5 Add a mixture of lentivirus particles obtained in step S2, transfection aids, and complete culture medium, mixed thoroughly in a ratio of 10:86:4, to a plate containing MSCs, and incubate for 48 hours.

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