A genetic engineering method for constructing and applying anti-aging human mesenchymal stem cells

Abstract

This invention belongs to the field of genetic engineering and cell modification technology, and discloses a method for constructing and applying anti-aging human mesenchymal stem cells (MSCs). The DHX9-overexpressing virus-transfected MSCs constructed in this invention possess stable DHX9 overexpression effects and anti-aging functions. These MSCs do not target a single gene locus, but rather alleviate a series of aging phenotypes and genomic instability issues during the aging process by eliminating R-loops. DHX9-overexpressing MSCs exhibit significant anti-aging activity, as well as strong environmental adaptability and excellent safety characteristics. They can effectively resist harsh in vivo inflammation and the aging microenvironment, while avoiding the tumorigenic risk after cell transplantation. These MSCs with stable anti-aging capabilities can subsequently be used to significantly improve the efficacy of MSC infusion therapy for diseases.

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] Current technical approaches for anti-aging human mesenchymal stem cells include autophagy activators, antioxidants, metabolic interventions, and genetic engineering. Autophagy activators include rapamycin; antioxidants include ascorbic acid and N-acetyl-L-cysteine; metabolic interventions include supplementing α-ketoglutarate (AKG) or activating isocitrate dehydrogenase (IDH) (such as ligustrazine); and genetic engineering includes overexpressing ERBB4 to enhance antioxidant capacity and overexpressing FOXO3 to enhance expression and construct anti-aging mesenchymal stem cells (SRCs).

[0004] 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. While current antioxidants (such as vitamin C) or autophagy activators (such as rapamycin) can reverse MSC senescence to some extent in vitro, the complex in vivo microenvironment, including factors like oxidative stress and chronic inflammation, can alter the effects of these microenvironments, 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 lifespan 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 involved in cellular senescence make it difficult to alter overall cellular function through single-gene editing.

[0005] Therefore, this invention provides a genetic engineering method for constructing and applying anti-aging human mesenchymal stem cells. Summary of the Invention

[0006] The purpose of this invention is to provide a genetic engineering construction method and application of anti-aging human mesenchymal stem cells. Based on eliminating the R-loop formed during cell aging from the source and alleviating the adverse effects of genomic instability caused by the R-loop, a DHX9 overexpression virus transfected MSCs targeting the elimination of cell R-loop has been developed, which solves the technical problem of the difficulty in constructing stable and effective MSCs with anti-aging ability after in vivo infusion in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, the present invention provides the application of genetically engineered human mesenchymal stem cells overexpressing DHX9 in the preparation of anti-aging and / or anti-inflammatory drugs.

[0009] On the one hand, the present invention also provides a method for constructing the above-mentioned genetically engineered human mesenchymal stem cells overexpressing DHX9, comprising the following steps:

[0010] S1. Insert the target gene into a lentiviral system vector to construct the GV492-DHX9 recombinant plasmid. The amino acid sequence of the target gene is shown in SEQ ID NO.1.

[0011] S2. The GV492-DHX9 recombinant plasmid obtained in S1 and the packaging plasmid were co-transfected into host cells to prepare lentiviral particles.

[0012] S3. Transfect human mesenchymal stem cells with the lentiviral particles obtained in S2 to obtain genetically engineered human mesenchymal stem cells overexpressing DHX9.

[0013] Preferably, the target gene is DHX9, which is obtained by using a forward primer: 5'-CGAACCATCTCAGCGACAAAA-3' and a reverse primer: 5'-TGAGGTCCATGCTTATT.

[0014] TGCTC-3' was cloned after PCR amplification.

[0015] Preferably, the lentiviral system vector is GV492, specifically pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.

[0016] Preferably, the host cell is a 293T cell.

[0017] Preferably, the packaging plasmids are psPAX2 and pMD2.G.

[0018] Preferably, in step 3, the transfection specifically involves: in a solution containing 1 x 10 5 Add a mixture of DHX9 overexpression lentivirus, transfection aid Polybrene, and complete culture medium DMEM to each well of MSCs in a ratio of 10:48:4, and incubate for 48 hours.

[0019] On the one hand, the present invention also provides genetically engineered human mesenchymal stem cells overexpressing DHX9 prepared by the above construction method.

[0020] On the one hand, the present invention also provides the application of the above-mentioned GV492-DHX9 recombinant plasmid or lentiviral particles in the preparation of anti-aging and / or anti-inflammatory drugs.

[0021] On the one hand, the present invention also provides the application of R-loop as a target of DHX9 gene action in the preparation of anti-aging and / or anti-inflammatory drugs.

[0022] Preferably, the drug comprises overexpressing DHX9 or silencing the R-loop.

[0023] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0024] Preferably, the drug is one that upregulates DHX9, ATP, and OCR levels, downregulates R-loop levels, and downregulates SASP molecules IL-1A, IL-1B, CCL2, CCL5, IL-6, and TGF-β levels.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention utilizes a strategy of stably overexpressing DHX9 via lentivirus to target the elimination of genomic R-loop structures, thereby developing anti-aging human mesenchymal stem cells (MSCs). Compared to existing approaches, this method is the first to propose a strategy of stably overexpressing DHX9 to eliminate genomic R-loop structures, enhancing the anti-aging ability of MSCs after in vivo infusion. This strategy does not target a specific gene, but rather focuses on the core mechanism mediating cellular senescence—the exacerbation of genomic instability. It constructs anti-aging MSCs by overexpressing DHX9 to eliminate the important culprit of this instability—the R-loop structure. Stably overexpressing DHX9-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, thus stably exerting their therapeutic effect and improving in vivo MSC infusion therapy. Attached Figure Description

[0027] 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 MSCs passed down from young passages to the 12th generation.

[0028] Figure 2 It is an immunoblotting and quantitative analysis of P16 and P21 protein expression in MSCs from different groups using Western blotting.

[0029] Figure 3 These are the results of RT-qPCR detection of the expression levels of P16 and P21 mRNA in MSCs from different groups;

[0030] 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;

[0031] 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;

[0032] Figure 6These are the results of ATP level detection in different groups of MSCs;

[0033] Figure 7 These are the results of OCR level detection for different groups of MSCs;

[0034] 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.

[0035] Figure 9 It is an immunoblot chromatogram and quantitative analysis of R-loop levels of MSCs in different groups using S9.6 Dot blot;

[0036] Figure 10 The results are from transcriptome sequencing of DHX9 expression levels in different groups of MSCs;

[0037] Figure 11 This is a flowchart of a mouse model of CIA arthritis treated with OE-DHX9-MSCs (DHX9 overexpressing MSCs) infusion;

[0038] Figure 12 It is an immunoblotting and quantitative analysis of DHX9 protein expression in MSCs from different groups using Western blotting.

[0039] Figure 13 These are the results of RT-qPCR detection of DHX9 mRNA expression levels in different groups of MSCs;

[0040] Figure 14 This is an immunoblotting and quantitative analysis of DHX9 protein expression in OE-DHX9 group MSCs detected by Western blotting (Vector refers to empty vector, OE-DHX9 refers to DHX9 overexpressing viral vector).

[0041] Figure 15 This is an S9.6 immunofluorescence assay showing the fluorescence pattern and quantitative analysis of R-loop levels in DHX9-overexpressing MSCs from different groups (elderly refers to elderly primary MSCs, elderly +OE-DHX9 refers to elderly primary MSCs overexpressing DHX9, UV...). + "P12" refers to naturally aging MSCs passaged to the 12th generation after UV-induced aging; "P12+OE-DHX9" refers to naturally aging MSCs passaged to the 12th generation after UV-induced aging and overexpressing DHX9.

[0042] Figure 16This is an immunoblot and quantitative analysis of the R-loop levels of DHX9 MSCs overexpressing in different groups, detected by S9.6 Dot blot.

[0043] Figure 17 This is a staining pattern and quantitative analysis of β-galactosidase staining to detect the senescence level of DHX9 overexpressing MSCs in different groups;

[0044] Figure 18 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 DHX9 MSCs overexpressing in different groups;

[0045] Figure 19 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 overexpressing DHX9 MSCs;

[0046] Figure 20 These are the ATP levels detected in DHX9-overexpressing MSCs from different groups;

[0047] Figure 21 These are the OCR levels of DHX9 MSCs overexpressing in different groups;

[0048] Figure 22 These are gross images of the ankle joint of mice after OE-DHX9-MSCs tail vein infusion treatment, along with tissue HE staining and Safranin-Fix Green staining results.

[0049] Figure 23 These are the results of arthritis scores and palm thickness measurements in mice after OE-DHX9-MSCs treatment. Detailed Implementation

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] 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.

[0053] Example 1: Extraction of MSCs to construct a senescent cell model

[0054] 1. Experimental Methods

[0055] 1) Construction of senescent cell model

[0056] Construction of primary senescent cell models: 30 ml of bone marrow was extracted from the posterior superior iliac spine of both elderly and young healthy volunteers, 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 to obtain elderly and young primary MSCs. The elderly and young primary MSCs were passaged to the 3rd generation, respectively, at a ratio of 1 × 10⁻⁶ cells / mL. 5 / wells were seeded in 6-well plates to obtain aged primary MSCs (referred to as aged group) and young primary MSCs (referred to as young group).

[0057] 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 was seeded in 6-well plates to obtain young passaged naturally senescent MSCs (denoted as P12 group).

[0058] 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 / wells 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, the cells were cultured for 72 hours to obtain UV-induced young passaged MSCs (denoted as the UV+ group).

[0059] 2) Measure various indicators within the senescent cell models MSCs constructed in 1).

[0060] 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.

[0061] 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.

[0062] 2. Experimental Results

[0063] 2.1 Senescence levels in aged and young MSCs stained with β-galactosidase

[0064] 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 ).

[0065] 2.2 Western Blot and RT-qPCR detection of P16 / P21 expression in aged and young MSCs

[0066] 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 ).

[0067] 2.3 RT-qPCR and ELISA detection of SASP expression and secretion in aged and young MSCs

[0068] 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 ).

[0069] 2.4 ATP Level Detection: ATP levels in aged and young MSCs

[0070] 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 ).

[0071] 2.5 OCR Level Detection of Oxidative Respiration Levels in Aged and Young MSCs

[0072] 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 ).

[0073] 2.6 S9.6 immunofluorescence and S9.6 dot blot detection of R-loop levels in aged and young MSCs

[0074] 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 ).

[0075] 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.

[0076] Example 2: Construction and application of DHX9 overexpression lentivirus

[0077] Previous research in this invention found that R-loops accumulate abnormally in senescent MSCs, and that this abnormal accumulation mediates genomic instability, leading to cell cycle arrest and cellular senescence. This embodiment, through transcriptome sequencing data, found that the TPM value of DHX9 was significantly reduced in both young and aged MSCs, indicating that the RNA helicase protein DHX9 was significantly downregulated in aged mesenchymal stem cells (see...). Figure 10 In other words, DHX9 downregulation mediates the accumulation of genomic R-loop. Therefore, a DHX9-overexpressing lentivirus was constructed to verify the effect of DHX9 overexpression on the anti-aging effect of mesenchymal stem cells. DHX9-overexpressing MSCs with stable anti-aging ability were used for infusion therapy in CIA arthritis model mice, and good therapeutic effects were achieved.

[0078] I. Construction of DHX9 overexpression lentivirus

[0079] (1) Plasmid construction:

[0080] The human DHX9 gene (NM_001357) was obtained from the cDNA library of Genechem (Shanghai Genechem Medical Technology Co., Ltd.) using the following primers: DHX9 forward primer: 5'-CGAACCATCTCAGCGACAAAA-3', DHX9 reverse primer: 5'-TGAGGTCCATGCTTATTTGCTC-3'; the DHX9 gene with the cloned amino acid sequence is shown in SEQ ID NO.1.

[0081] The lentiviral vector plasmid GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin) (purchased from Shanghai Jikai Gene Medical Technology Co., Ltd.) and the DHX9 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.

[0082] The amino acid sequence of DHX9 (SEQ ID NO.1) is as follows:

[0083]

[0084] (2) Lentiviral production:

[0085] 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.

[0086] 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.

[0087] II. Application of DHX9 overexpression lentiviruses

[0088] 1. Experimental Methods

[0089] (1) In vitro cell experiments

[0090] 1) DHX9-overexpressing lentivirus was used to transfect aged primary MSCs, young naturally senescent MSCs passaged to the 12th generation, and UV-induced senescent passaged MSCs prepared in Example 1 for 48 hours, as follows: 1 x 10 5 After seeding MSCs into 12-well plates, aspirate the supernatant, mix DHX9 overexpression lentivirus, transfection aid Polybrene, and complete culture medium in a DMEM ratio of 10:48:4 thoroughly, add 2 ml / well, and incubate for 48 hours.

[0091] 2) DHX9 protein expression in MSCs was detected using Western blot, and DHX9 mRNA expression in MSCs was detected using RT-qPCR. Intracellular R-loop levels in MSCs were detected using immunofluorescence staining and Dot blot. MSC senescence levels were detected using a β-galactosidase staining kit. P16 / P21 protein expression in MSCs was detected using Western blot. P16 / P21 mRNA expression in MSCs was detected using RT-qPCR. SASP mRNA expression in MSCs was detected using RT-qPCR, and SASP secretion in MSCs was detected using an ELISA kit. ATP levels in MSCs were detected using an ATP assay kit. OCR levels in MSCs were detected using an OCR assay kit.

[0092] (2) In vivo animal experiments

[0093] 1) Construct a mouse model of CIA arthritis. The specific construction plan is as follows:

[0094] On day 1, male DBA / 1 mice (6-8 weeks old, weighing 20-30 g) were subcutaneously injected with 100 μL of a 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 100 μL of an 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.

[0095] Arthritis severity assessment:

[0096] 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.

[0097] Hind paw thickness measurement: The thickness of the mouse's hind paw was measured using vernier calipers to assess the severity of inflammation.

[0098] 2) OE-DHX9-MSCs infusion therapy in a mouse model of CIA arthritis

[0099] For detailed procedures, please see [link / document / documentation]. Figure 11 After 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.

[0100] 2. Experimental Results

[0101] (1) In vitro cell experiments

[0102] Western blot and RT-qPCR assays showed that DHX9 protein levels were significantly downregulated in aged MSCs: Compared to younger MSCs, aged MSCs had lower levels of both DHX9 protein and mRNA; compared to the UV- group, UV+ group MSCs had lower levels of both DHX9 protein and mRNA; compared to the P3 group, P12 group MSCs had lower levels of both DHX9 protein and mRNA (see [link to data]). Figure 12 and Figure 13 ).

[0103] Western blot analysis showed that DHX9 protein was overexpressed in the OE-DHX9 group: compared to the Vector group MSCs, the OE-DHX9 group MSCs showed higher DHX9 protein levels through immunoblotting and quantification (see [link to data]). Figure 14 ).

[0104] S9.6 Immunofluorescence and Dot blot analysis showed that the R-loop level of DHX9-overexpressing MSCs was significantly reduced: The R-loop level was lower in the elderly + OE-DHX9 group compared to the elderly group; the R-loop level was lower in the UV + OE-DHX9 group compared to the UV + group; and the R-loop level was lower in the P12 + OE-DHX9 group compared to the P12 group (see [link to study].) Figure 15 and Figure 16 ).

[0105] β-galactosidase staining showed that senescence levels were significantly reduced in DHX9-overexpressing MSCs: compared to the aged group MSCs, the aged + OE-DHX9 group MSCs stained lighter and had a lower proportion of positive cells; compared to the UV+ group MSCs, the UV+OE-DHX9 group MSCs stained lighter and had a lower proportion of positive cells; compared to the P12 group MSCs, the P12+OE-DHX9 group MSCs stained lighter and had a lower proportion of positive cells (see...). Figure 17 ).

[0106] RT-qPCR and ELISA showed significantly reduced SASP expression and secretion levels in DHX9-overexpressing MSCs: Compared to the elderly group MSCs, the elderly + OE-DHX9 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-DHX9 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-DHX9 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 18 and Figure 19 ).

[0107] ATP assays showed significantly elevated ATP levels in MSCs overexpressing DHX9: MSCs in the aged + OE-DHX9 group had higher ATP levels than those in the aged group; MSCs in the UV + OE-DHX9 group had higher ATP levels than those in the UV + group; and MSCs in the P12 + OE-DHX9 group had higher ATP levels than those in the P12 group (see [link to study].) Figure 20 ).

[0108] OCR detection showed significantly elevated OCR levels in DHX9-overexpressing MSCs: The OCR levels were higher in the elderly + OE-DHX9 group compared to the elderly group; higher in the UV + OE-DHX9 group compared to the UV + group; and higher in the P12 + OE-DHX9 group compared to the P12 group (see [link to OCR analysis]). Figure 21 ).

[0109] In summary, overexpression of DHX9 significantly upregulated DHX9 mRNA and protein levels, significantly downregulated R-loop levels, reduced 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 DHX9 can significantly promote the anti-aging ability of MSCs.

[0110] (2) In vivo animal experiments

[0111] OE-DHX9-MSCs infusion treatment of CIA arthritis mouse model, gross images of the ankle joint, tissue HE staining, Safranin-Fix-Green staining: Compared with the CIA group and the CIA+MSCs infusion treatment group, the CIA+OE-DHX9-MSCs infusion treatment group mice had less ankle joint swelling, less synovial hyperplasia, less inflammatory cell infiltration, and less joint damage.

[0112] OE-DHX9-MSCs infusion treatment of CIA arthritis mouse model, arthritis score and palm thickness measurement: Compared with the CIA group and the CIA+MSCs infusion treatment group, the CIA+OE-DHX9-MSCs infusion treatment group mice had lower arthritis scores and thinner palms.

[0113] In summary, OE-DHX9-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.

[0114] Therefore, the DHX9-overexpressing virus-transfected MSCs constructed in this invention possess stable DHX9 overexpression effects and anti-aging functions. These MSCs do not target a single gene locus but alleviate a series of aging phenotypes and genomic instability issues during the aging process by eliminating R-loops. DHX9-overexpressing MSCs exhibit significant anti-aging activity, strong environmental adaptability, and excellent safety profile. They can also 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 be subsequently applied to significantly improve the efficacy of MSC infusion therapy for diseases.

[0115] 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. The application of human mesenchymal stem cells overexpressing DHX9 in the preparation of anti-aging or anti-arthritis drugs, characterized in that, The amino acid sequence of DHX9 is shown in SEQ ID NO.1, and the human mesenchymal stem cells are derived from bone marrow.

2. The application according to claim 1, characterized in that, The method for constructing human mesenchymal stem cells overexpressing DHX9 includes the following steps: S1. Insert the target gene into a lentiviral system vector to construct the GV492-DHX9 recombinant plasmid, wherein the target gene is DHX9; S2. The GV492-DHX9 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 genetically engineered human mesenchymal stem cells overexpressing DHX9.

3. The application according to claim 2, characterized in that, The DHX9 was obtained by cloning after PCR amplification using forward primer: 5'-CGAACCATCTCAGCGACAAAA-3' and reverse primer: 5'-TGAGGTCCATGCTTATTTGCTC-3'.

4. The application according to claim 2, characterized in that, The lentiviral system vector is pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.

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

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

7. The application according to claim 2, characterized in that, In step 3, the transfection specifically involves: in a solution containing 1 x 10 5 Add a mixture of DHX9 overexpression lentivirus, transfection aids, and complete culture medium in a ratio of 10:48:4 to each well of MSCs and incubate for 48 hours.

8. The application according to claim 1, characterized in that, The drug comprises human mesenchymal stem cells overexpressing DHX9 and a pharmaceutically acceptable vector.

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