Genetic engineering construction method and application of anti-aging human mesenchymal stem cells

Abstract

The invention belongs to the technical field of gene engineering and cell transformation, and discloses a gene engineering construction method and application of anti-aging human mesenchymal stem cells. The DHX9 overexpression virus transfected MSCs constructed by the invention have a stable DHX9 overexpression effect and an anti-aging function, and the MSCs relieve a series of problems of aging phenotypes and unstable genomes in an aging process by eliminating R-loop instead of aiming at a single gene locus. The DHX9 overexpression MSCs show significant anti-aging activity, also have strong adaptive capacity to environment and excellent safety characteristics, can effectively resist harsh in-vivo inflammation and aging microenvironment, and at the same time avoids tumorigenic risk after cell transplantation. The MSCs with the stable anti-aging capability can be subsequently applied to remarkably improving the treatment effect of MSCs infused into a body for treating diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and cell modification, and relates to a construction method of mesenchymal stem cells, in particular to a genetic engineering construction method of anti-aging human mesenchymal stem cells and application. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) belong to multipotent adult stem cells of mesoderm origin, which exist in bone marrow, fat, umbilical cord, placenta, dental pulp, synovial membrane and other tissues; can be long-term expanded in vitro, and can differentiate into bone, cartilage, fat, nerve, myocardial and liver cells and other cell types, do not express HLA-DR antigen (human leukocyte antigen-DR), only weakly express HLA-A / B (human leukocyte antigen A / B), do not need strict matching for allogeneic transplantation, and rarely cause immune rejection; can regulate microenvironment and promote tissue regeneration through chemotaxis and homing effect, and secretion effect. Mesenchymal stem cell infusion therapy shows significant potential in the treatment of many diseases, and has been used for systemic lupus erythematosus, rheumatoid arthritis and other autoimmune diseases; osteoarthritis and other motor system diseases; type II diabetes and other endocrine system diseases; acute myocardial infarction, heart failure and other circulatory system diseases; spinal cord injury and brain injury and other nervous system diseases. Aging of MSCs refers to the biological process that, under physiological or pathological conditions, due to cumulative damage, the self-renewal ability, multilineage differentiation potential and paracrine function gradually decline. This process is accompanied by characteristic changes in cell morphology, gene expression and metabolic activity, and ultimately results in the loss of repair and regeneration ability, which is one of the important mechanisms of whole body aging and the occurrence of aging-related diseases.

[0003] The current technical solutions of anti-aging human mesenchymal stem cells include autophagy activators, antioxidants, metabolic intervention and genetic engineering. Autophagy activators include rapamycin and the like; antioxidants include ascorbic acid, N-acetyl-L-cysteine; metabolic intervention includes supplementing alpha-ketoglutarate (AKG) or activating isocitrate dehydrogenase (IDH) (such as scullcap ethyl); genetic engineering includes overexpression of ERBB4 to enhance antioxidant capacity, overexpression of FOXO3 to enhance expression of anti-aging mesenchymal stem cells (SRC), and the like.

[0004] Although good progress has been made in the study of mesenchymal stem cell aging in recent years, the mechanism of cell aging is very complex, and the exact cause of cell aging cannot be determined at present, and a method for targeted intervention in mesenchymal stem cell aging cannot be found. Although the current antioxidants (such as vitamin C) or autophagy activators (such as rapamycin) can reverse MSCs aging to a certain extent in vitro, the microenvironment in vivo is complex, such as oxidative stress, chronic inflammation and other microenvironments. After the cells enter the body microenvironment, they may be affected by the microenvironment and change, resulting in a significant decrease in effect. Furthermore, the in vitro culture conditions cannot simulate the dynamic changes of the in vivo aging microenvironment, such as the gradient of inflammatory factors and the interference of cell-cell communication. At the same time, the in vitro cell intervention based on drugs has a certain timeliness and cannot achieve stable anti-aging effect. The current gene engineering often overexpresses or knocks out a single gene site, but the multi-gene interaction network in the cell aging process is complex, and single editing cannot change the overall function of the cell.

[0005] Therefore, the application provides a gene engineering construction method and application of an anti-aging human mesenchymal stem cell. SUMMARY

[0006] The purpose of the application is to provide a gene engineering construction method and application of an anti-aging human mesenchymal stem cell, which eliminates R-loop (R-loop) formed in the cell aging process from the source, alleviates the adverse effects of genomic instability caused by R-loop, and develops DHX9 overexpression virus transfection MSCs for cell R-loop elimination, solving the technical problem that it is difficult to construct stable and effective MSCs with anti-aging ability after in vivo infusion in the prior art.

[0007] In order to achieve the above purpose, the application adopts the following technical scheme:

[0008] On the one hand, the application provides a use of a gene engineered DHX9 overexpression human mesenchymal stem cell in the preparation of an anti-aging and / or anti-inflammatory drug.

[0009] On the one hand, the application also provides a construction method of the above-mentioned gene engineered DHX9 overexpression human mesenchymal stem cell, comprising the following steps:

[0010] S1, inserting a target gene into a lentivirus system vector to construct a GV492-DHX9 recombinant plasmid, the amino acid sequence of the target gene is shown as SEQ ID NO. 1;

[0011] S2, co-transfecting the GV492-DHX9 recombinant plasmid obtained in S1 and a packaging plasmid into a host cell to prepare a lentivirus particle;

[0012] S3, transfecting the lentivirus particles obtained in S2 into human mesenchymal stem cells to obtain genetically engineered human mesenchymal stem cells overexpressing DHX9.

[0013] Preferably, the target gene is DHX9, which is cloned after PCR amplification using the forward primer 5'-CGAACCATCTCAGCGACAAAA-3' and the reverse primer 5'-TGAGGTCCATGCTTATT TGCTC-3'.

[0014] Preferably, the lentivirus system vector is GV492, specifically pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.

[0015] Preferably, the host cell is a 293T cell.

[0016] Preferably, the packaging plasmid is psPAX2 and pMD2.G.

[0017] Preferably, in step 3, the transfection is specifically: adding a mixture of sufficient DHX9 overexpression lentivirus, transfection auxiliary reagent Polybrene and complete culture medium DMEM in a ratio of 10:48:4 into a well plate containing 1 x 10 5 MSCs, and culturing for 48 hours.

[0018] In one aspect, the present application also provides genetically engineered human mesenchymal stem cells overexpressing DHX9 prepared by the above construction method.

[0019] In one aspect, the present application also provides the use of the above GV492-DHX9 recombinant plasmid or lentivirus particles in the preparation of anti-aging and / or anti-inflammatory drugs.

[0020] In one aspect, the present application also provides the use of R-loop as a target of DHX9 gene in the preparation of anti-aging and / or anti-inflammatory drugs.

[0021] Preferably, the drug includes overexpression of DHX9 or silencing of R-loop.

[0022] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0023] Preferably, the drug is a drug that upregulates the levels of DHX9, ATP and OCR, downregulates the level of R-loop, and downregulates the levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] The application is a strategy of stably overexpressing DHX9 by lentivirus to develop anti-aging human mesenchymal stem cells by targeting genome R-loop. Compared with the existing scheme, the present scheme first proposes a strategy of stably overexpressing DHX9 to clear the genome R-loop structure, thereby improving the anti-aging ability of MSCs after being injected into the body. The above strategy does not target a certain gene, but targets the core mechanism of cell aging, i.e., the aggravation of genomic instability, and constructs anti-aging MSCs by overexpressing DHX9 to clear the important "perpetrator" of the aggravation of genomic instability, i.e., the R-loop structure. The stably overexpressing DHX9-MSCs is expected to stably clear the R-loop structure in the cells, better cope with various stimuli inducing cell aging when facing the complex in vivo microenvironment after being injected into the body, thereby stably exerting the therapeutic effect and improving the MSCs in vivo injection treatment scheme. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a staining chart and quantitative analysis of β-galactosidase staining for detecting the aging level of MSCs in different groups (young refers to young primary MSCs, old refers to old primary MSCs, UV - refers to young passage 3 MSCs without ultraviolet-induced aging, UV + refers to young passage 3 MSCs with ultraviolet-induced aging, P3 refers to young passage 3 MSCs, and P12 refers to naturally aged MSCs of young passage to the 12th generation);

[0027] Figure 2 is an immunoblotting chart and quantitative analysis of Western Blot for detecting the P16 and P21 protein expression of MSCs in different groups;

[0028] Figure 3 is the result of RT-qPCR for detecting the P16 and P21 mRNA expression level of MSCs in different groups;

[0029] Figure 4 is the result of RT-qPCR for detecting the mRNA expression level of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β of MSCs in different groups;

[0030] Figure 5 is the result of ELISA for detecting the protein secretion level of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β of MSCs in different groups;

[0031] Figure 6 is the result of ATP level detection of MSCs in different groups;

[0032] Figure 7 are the results of OCR level detection of MSCs in different groups;

[0033] Figure 8 It is the fluorescence image and quantitative analysis of R-loop levels in MSCs of different groups detected by S9.6 immunofluorescence;

[0034] Figure 9 It is the immunoblot image and quantitative analysis of the R-loop level of MSCs in different groups detected by S9.6 Dot blot;

[0035] Figure 10 is the result of transcriptome sequencing of DHX9 expression levels in MSCs of different groups;

[0036] Figure 11 This is a flow chart of the OE-DHX9-MSCs (DHX9 overexpressing MSCs) infusion therapy for the CIA arthritis mouse model;

[0037] Figure 12 It is the immunoblot image and quantitative analysis of DHX9 protein expression in MSCs of different groups detected by Western Blot;

[0038] Figure 13 This is the result of RT-qPCR detection of DHX9 mRNA expression levels in MSCs of different groups;

[0039] Figure 14 This is the immunoblot image and quantitative analysis of DHX9 protein expression in MSCs of the OE-DHX9 group detected by Western Blot (Vector refers to empty vector, and OE-DHX9 refers to DHX9 overexpressing viral vector);

[0040] Figure 15 The fluorescence images and quantitative analysis of R-loop levels in MSCs overexpressing DHX9 in different groups were detected by S9.6 immunofluorescence (Aged refers to aged primary MSCs, Aged+OE-DHX9 refers to aged primary MSCs overexpressing DHX9, UV + refers to the third passage of MSCs induced by UV senescence, UV+OE-DHX9 refers to the third passage of MSCs induced by UV senescence with overexpression of DHX9, P12 refers to naturally senescent MSCs that were passaged to the 12th passage from young age, and P12+OE-DHX9 refers to naturally senescent MSCs that were passaged to the 12th passage from young age with overexpression of DHX9);

[0041] Figure 16Figure 9.6 is an immunoblotting diagram and quantitative analysis of R-loop levels of MSCs overexpressing DHX9 in different groups detected by S9.6 Dot blot;

[0042] Figure 17 Figure 9.4 is a staining diagram and quantitative analysis of the senescence level of MSCs overexpressing DHX9 in different groups detected by β-galactosidase staining;

[0043] Figure 18 Figure 9.7 is the result of RT-qPCR detection of the mRNA expression levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β of MSCs overexpressing DHX9 in different groups;

[0044] Figure 19 Figure 9.8 is the result of ELISA detection of the protein secretion levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β of MSCs overexpressing DHX9 in different groups;

[0045] Figure 20 Figure 9.9 is the result of detection of ATP levels of MSCs overexpressing DHX9 in different groups;

[0046] Figure 21 Figure 9.10 is the result of detection of OCR levels of MSCs overexpressing DHX9 in different groups;

[0047] Figure 22 Figure 9.11 is the result of local gross image, tissue HE staining, and ponceau-fuchsin staining of the ankle joint of mice after OE-DHX9-MSCs tail vein infusion treatment;

[0048] Figure 23 Figure 9.12 is the result of arthritis score and palm thickness measurement of mice after OE-DHX9-MSCs treatment. DETAILED DESCRIPTION

[0049] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0050] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0051] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0052] Example 1 Extraction of MSCs to construct a senescent cell model

[0053] 1. Experimental methods

[0054] 1) Construction of aging cell models

[0055] Construction of primary aging cell models: 30 ml of bone marrow was extracted from an elderly healthy volunteer and a young healthy volunteer respectively via the posterior superior iliac spine, and was anticoagulated with heparin. The bone marrow was mixed with PBS and centrifuged (1500 rpm, 5 min) to remove the supernatant and fat. The cell suspension was slowly added to the upper layer of lymphocyte separation medium and centrifuged (2000 rpm, 20 min); the white membrane layer containing MSCs was aspirated. PBS was used for washing twice, and the cells were resuspended and inoculated in a culture medium containing 15% FBS for culture, to obtain elderly primary MSCs and young primary MSCs. The elderly primary MSCs and young primary MSCs were subcultured to the 3rd generation, and were inoculated in a 6-well plate at a density of 1 x 10 5 / well to obtain elderly primary MSCs (referred to as the elderly group) and young primary MSCs (referred to as the young group).

[0056] Construction of subcultured aging cell models: the young MSCs were subcultured to the 12th generation, and were inoculated in a 6-well plate at a density of 1 x 10 5 / well to obtain young subcultured naturally aging MSCs (referred to as the P12 group).

[0057] Construction of ultraviolet-induced aging cell models: the young primary MSCs were subcultured to the 3rd generation, and were inoculated in a 6-well plate at a density of 1 x 10 5 / well. After a single irradiation of UVB (medium wave ultraviolet, 280-320 nm) at 40 mJ / cm², the young subcultured MSCs were cultured for 72 hours to obtain ultraviolet-induced young subcultured MSCs (referred to as the UV+ group).

[0058] 2) Determination of various indicators in the MSCs of the aging cell models constructed in 1)

[0059] The β-galactosidase staining kit was used to detect the aging level of the MSCs, Western blot was used to detect the P16 / P21 protein expression of the MSCs, and RT-qPCR was used to detect the P16 / P21 mRNA expression of the MSCs;

[0060] RT-qPCR was used to detect the SASP mRNA expression of the MSCs, ELISA kit was used to detect the SASP secretion of the MSCs, ATP detection kit was used to detect the ATP level of the MSCs, OCR detection kit was used to detect the OCR (oxygen consumption rate) level of the MSCs, and immunofluorescence staining and Dot blot were used to detect the R-loop level in the MSCs.

[0061] 2. Experimental results

[0062] 2.1 β-galactosidase staining of the aging levels of elderly and young MSCs

[0063] The results of β-galactosidase staining showed that compared with MSCs in the young group, MSCs in the elderly group had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells; compared with MSCs in the UV- group, MSCs in the UV+ group had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells; compared with MSCs in the P3 group, MSCs in the P12 group had darker β-galactosidase staining and a higher proportion of β-galactosidase-positive cells (see Figure 1 ).

[0064] 2.2 Western Blot and RT-qPCR detection of P16 / P21 expression in aged and young MSCs

[0065] Western Blot and RT-qPCR results showed that the expression levels of P16 and P21 proteins and mRNA in MSCs from the old group were higher than those in MSCs from the young group; the expression levels of P16 and P21 proteins and mRNA in MSCs from the UV+ group were higher than those in MSCs from the UV- group; and the expression levels of P16 and P21 proteins and mRNA in MSCs from the P12 group were higher than those in MSCs from the P3 group (see Figure 2 and Figure 3 ).

[0066] 2.3 Detection of SASP expression and secretion in aged and young MSCs by RT-qPCR and ELISA

[0067] Compared with MSCs in the young group, the mRNA expression levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6, and TGF-β in MSCs in the elderly group were higher, and the protein secretion level was higher. Compared with MSCs in the UV- group, the mRNA expression levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6, and TGF-β in MSCs in the UV+ group were higher, and the protein secretion level was higher. Compared with MSCs in the P3 group, the mRNA expression levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6, and TGF-β in MSCs in the P12 group were higher, and the protein secretion level was higher ( Figure 4 and Figure 5 ).

[0068] 2.4 ATP level detection ATP level in old and young MSCs

[0069] Compared with the young group MSCs, the ATP level of the old group MSCs was lower; compared with the UV- group MSCs, the ATP level of the UV+ group MSCs was lower; compared with the P3 group MSCs, the ATP level of the P12 group MSCs was lower (see Figure 6 ).

[0070] 2.5 OCR level detection of oxidative respiratory level of old and young MSCs

[0071] The OCR level of the old group MSCs is lower than that of the relatively young group MSCs; the OCR level of the UV+ group MSCs is lower than that of the UV- group MSCs; the OCR level of the P12 group MSCs is lower than that of the P3 group MSCs (see Figure 7 ).

[0072] 2.6 S9.6 immunofluorescence and S9.6 Dot blot detection of R-loop level of old and young MSCs

[0073] The results all show that the R-loop level of the old group MSCs is higher than that of the relatively young group MSCs; the R-loop level of the UV+ group MSCs is higher than that of the UV- group MSCs; the R-loop level of the P12 group MSCs is higher than that of the P3 group MSCs (see Figure 8 and Figure 9 ).

[0074] In summary, the R-loop level of the senescent MSCs is increased, the β-galactosidase staining quantification is high, the P16 / P21 protein and mRNA levels are high, the SASP expression and secretion levels are high, and the ATP and OCR levels are low.

[0075] Example 2 Construction and application of DHX9 overexpression lentivirus

[0076] The previous research of the present application found that R-loop abnormally accumulates in senescent MSCs, and the abnormal accumulation of R-loop mediates genomic instability, which in turn causes cell cycle arrest, resulting in cell senescence. In this embodiment, the transcriptome sequencing data found that the TPM value of DHX9 in the old group MSCs was significantly lower than that in the young group MSCs, that is, the RNA helicase protein DHX9 was significantly down-regulated in the old human mesenchymal stem cells (see Figure 10 ). That is, DHX9 down-regulation mediates the accumulation of genomic R-loop, so DHX9 overexpression lentivirus is constructed to verify the effect of overexpression of DHX9 on mesenchymal stem cells resisting aging, and the DHX9 overexpression MSCs with stable anti-aging ability are used for infusion treatment of CIA arthritis model mice, and good treatment effect is achieved.

[0077] I. Construction of DHX9 overexpression lentivirus

[0078] (1) Plasmid construction:

[0079] The human DHX9 gene (NM_001357) was obtained from a cDNA library from Genechem (Shanghai Jikai Genechem Biotech Co., Ltd.), using the following primers: DHX9 forward primer: 5'-CGAACCATCTCAGCGACAAAA-3', DHX9 reverse primer: 5'-TGAGGTCCATGCTTATTTGCTC-3'; the cloned DHX9 gene with the amino acid sequence shown in SEQ ID NO. 1.

[0080] The lentiviral vector plasmid GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin) (purchased from Shanghai Jikai Genechem Biotech Co., Ltd.) and the DHX9 gene sequence were respectively digested with Age I and Nhe I restriction endonucleases, and the cloning connection was completed by the In-fusion seamless cloning (recombination) method. The recombinant vector was identified by DNA sequencing detection.

[0081] The amino acid sequence of DHX9 (SEQ ID NO. 1) is:

[0082]

[0083] (2) Lentivirus production:

[0084] The above constructed viral vector and two kinds of auxiliary packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells using Lipofectamine 2000 (purchased from Invitrogen; Thermo Fisher Scientific, Inc.) together. The culture supernatant containing virus was collected 72 hours after transfection, and the cell debris was removed by rapid centrifugation at 1000xg, and then sterilized by 0.45 μm cellulose acetate filter membrane.

[0085] The virus titer was determined by flow cytometry (FACS) analysis of the proportion of GFP-positive 293T cells, about 1 x 10 9 Transduction units (TU) / mL of culture medium.

[0086] II. Application of DHX9 overexpression lentivirus

[0087] 1. Experimental method

[0088] (1) In vitro cell experiment

[0089] 1) DHX9 overexpression lentivirus was used to transfect the old primary MSCs prepared in Example 1, naturally senescent MSCs passaged to the 12th generation, and UV-induced senescent MSCs passaged to the 3rd generation for 48 hours, as follows: 1 x 10 5 After the MSCs were plated in a 12-well plate, the supernatant was removed, and the DHX9 overexpression lentivirus, transfection auxiliary reagent Polybrene and complete culture medium were mixed in a ratio of DMEM10:48:4, and then 2 ml / well was added and cultured for 48 hours.

[0090] 2) Western blot was used to detect the DHX9 protein expression of MSCs, RT-qPCR was used to detect the DHX9 mRNA expression of MSCs; immunofluorescence staining and Dot blot were used to detect the R-loop level in MSCs, β-galactosidase staining kit was used to detect the senescence level of MSCs, Western blot was used to detect the P16 / P21 protein expression of MSCs. RT-qPCR was used to detect the P16 / P21 mRNA expression of MSCs; RT-qPCR was used to detect the SASP mRNA expression of MSCs, ELISA kit was used to detect the SASP secretion of MSCs; ATP detection kit was used to detect the ATP level of MSCs. OCR detection kit was used to detect the OCR level of MSCs.

[0091] (2) In vivo animal experiments

[0092] 1) Construct CIA arthritic mouse model, the specific construction scheme is as follows:

[0093] On day 1, DBA / 1 male mice (6-8 weeks old, body weight 20-30 grams) were subcutaneously injected with 100 μL of a homogeneous emulsion mixture prepared by mixing chicken type II collagen (Chondrex, #20012) with an equal volume of Freund's complete adjuvant (Chondrex, #7001). 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 performed slowly into the tail root, and care was taken to avoid the area of visible large blood vessels at the site of the first injection.

[0094] Arthritis severity assessment:

[0095] Four limb arthritis score: Each leg (four limbs) of the mouse was scored for arthritis independently, and the scoring criteria were as follows: 0 points: normal; 1 point: single joint redness or swelling; 2 points: multiple joint redness or swelling; 3 points: entire foot palm redness or swelling; 4 points: joint ankylosis or severe deformity. The total arthritis score for each mouse was the sum of the scores for the four legs of the mouse.

[0096] Posterior paw thickness measurement: The thickness of the mouse's posterior paw was measured using a vernier caliper to assess the severity of inflammation.

[0097] 2) OE-DHX9-MSCs infusion treatment of CIA arthritic mouse model

[0098] The specific process is shown in Figure 11 , after successful construction, the local image of the mouse ankle joint was detected, HE staining, safranin-fast green staining, and arthritis score and palm thickness were measured.

[0099] 2. Experimental results

[0100] (1) In vitro cell experiment

[0101] Western Blot and RT-qPCR detection showed that the DHX9 protein level was significantly down-regulated in old MSCs: the DHX9 protein and mRNA levels of the old group MSCs were lower than those of the young group MSCs; the DHX9 protein and mRNA levels of the UV+ group MSCs were lower than those of the UV- group MSCs; the DHX9 protein and mRNA levels of the P12 group MSCs were lower than those of the P3 group MSCs (see Figure 12 and Figure 13 ).

[0102] Western Blot shows DHX9 protein level overexpression in OE-DHX9 group: relative to Vector group MSCs, OE-DHX9 group MSCs immunoblotting and quantification show higher DHX9 protein level (see Figure 14 ).

[0103] S9.6 immunofluorescence and Dot blot show significantly reduced R-loop level in overexpressing DHX9 MSCs: relative to old group MSCs, old + OE-DHX9 group MSCs have lower R-loop level; relative to UV+ group MSCs, UV+ OE-DHX9 group MSCs have lower R-loop level; relative to P12 group MSCs, P12 + OE-DHX9 group MSCs have lower R-loop level (see Figure 15 and Figure 16 ).

[0104] β-galactosidase staining shows significantly reduced senescence level in overexpressing DHX9 MSCs: relative to old group MSCs, old + OE-DHX9 group MSCs have lighter staining and less percentage of stained positive cells; relative to UV+ group MSCs, UV+ OE-DHX9 group MSCs have lighter staining and less percentage of stained positive cells; relative to P12 group MSCs, P12 + OE-DHX9 group MSCs have lighter staining and less percentage of stained positive cells (see Figure 17 ).

[0105] RT-qPCR and ELISA show significantly reduced SASP expression and secretion level in overexpressing DHX9 MSCs: relative to old group MSCs, old + OE-DHX9 group MSCs have lower mRNA expression and protein secretion level of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β; relative to UV+ group MSCs, UV+ OE-DHX9 group MSCs have lower mRNA expression and protein secretion level of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β; relative to P12 group MSCs, P12 + OE-DHX9 group MSCs have lower mRNA expression and protein secretion level of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β (see Figure 18 and Figure 19 ).

[0106] ATP detection The ATP level of MSCs overexpressing DHX9 was significantly increased: the ATP level of MSCs in the old + OE-DHX9 group was higher than that in the old group; the ATP level of MSCs in the UV+OE-DHX9 group was higher than that in the UV+ group; the ATP level of MSCs in the P12+OE-DHX9 group was higher than that in the P12 group (see Figure 20 ).

[0107] OCR detection The OCR level of MSCs overexpressing DHX9 was significantly increased: the OCR level of MSCs in the old + OE-DHX9 group was higher than that in the old group; the OCR level of MSCs in the UV+OE-DHX9 group was higher than that in the UV+ group; the OCR level of MSCs in the P12+OE-DHX9 group was higher than that in the P12 group (see Figure 21 ).

[0108] In summary, after overexpression of DHX9, the mRNA and protein levels of MSCs DHX9 were significantly increased, the R-loop level was significantly decreased, the β-galactosidase staining quantification was reduced, the P16 / P21 protein and mRNA levels were weakened, the SASP expression and secretion levels were weakened, and the ATP and OCR levels were increased, proving that overexpression of DHX9 can significantly promote the ability of MSCs to resist aging.

[0109] (2) In vivo animal experiments

[0110] OE-DHX9-MSCs infusion treatment of CIA arthritis mouse model, ankle joint local gross image, tissue HE staining, ponceau solid green staining: relative to CIA group and CIA+MSCs infusion treatment group mice, CIA+OE-DHX9 MSCs infusion treatment group mice ankle joint swelling degree is lower, synovial hyperplasia degree is less, inflammatory cell infiltration is less, joint destruction degree is lower.

[0111] OE-DHX9-MSCs infusion treatment of CIA arthritis mouse model, arthritis score and palm thickness measurement: relative to CIA group and CIA+MSCs infusion treatment group mice, CIA+OE-DHX9 MSCs infusion treatment group mice arthritis score is lower, palm thickness is thinner.

[0112] In summary, OE-DHX9-MSCs infusion treatment of CIA mouse arthritis model, joint local inflammation is relieved, arthritis score and palm thickness are reduced.

[0113] It can be seen that the DHX9 overexpression virus transfected MSCs constructed by the application has a stable DHX9 overexpression effect and anti-aging function, the MSCs are not aimed at a single gene site, but can relieve a series of aging phenotypes and genomic instability problems in the aging process by eliminating R-loop. The DHX9 overexpression MSCs exhibit significant anti-aging activity, have strong environmental adaptability and excellent safety characteristics, can effectively resist harsh in-vivo inflammation and aging microenvironment, and can avoid the tumorigenic risk after cell transplantation. The MSCs with stable anti-aging ability can be applied to significantly improve the curative effect of MSCs infusion in the treatment of diseases in-vivo.

[0114] The specific embodiments are only an explanation of the application, and are not a limitation of the application, and those skilled in the art can make modifications to the embodiments without creative contribution according to the needs after reading the specification, but as long as the modifications are within the scope of the claims of the application, they are protected by the patent law.

Claims

1. A method for constructing genetically engineered human mesenchymal stem cells overexpressing DHX9, characterized in that: The steps include: S1. Insert the target gene into the 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. S2, co-transfecting the GV492-DHX9 recombinant plasmid obtained in S1 with the packaging plasmid into host cells to prepare lentiviral particles; S3. Transfecting human mesenchymal stem cells with the lentiviral particles obtained in S2 to obtain genetically engineered human mesenchymal stem cells overexpressing DHX9; the human mesenchymal stem cells overexpressing DHX9 are used to prepare anti-aging and / or anti-inflammatory drugs.

2. The construction method according to claim 1, characterized in that The target gene is DHX9, which is cloned after PCR amplification using a forward primer: 5'CGAACCATCTCAGCGACAAAA-3' and a reverse primer: 5'-TGAGGTCCATGCTTATTTGCTC-3'.

3. The construction method according to claim 1, characterized in that The lentiviral system vector is pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.

4. The construction method according to claim 1, wherein The host cell is 293T cell.

5. The construction method according to claim 1, characterized in that The packaging plasmids are psPAX2 and pMD2.G.

6. The construction method according to claim 1, characterized in that In step 3, the transfection is specifically as follows: 5 A mixture of DHX9 overexpression lentivirus, transfection auxiliary reagent and complete culture medium in a ratio of 10:48:4 was added to a well plate containing MSCs and cultured for 48 hours. 7 . Human mesenchymal stem cells overexpressing DHX9 prepared by the construction method according to any one of claims 1 to 6 .

8. Use of the DHX9-overexpressing human mesenchymal stem cells according to claim 7 in the preparation of anti-aging and / or anti-inflammatory drugs.

9. Use of the GV492-DHX9 recombinant plasmid or lentiviral particle as claimed in claim 1 in the preparation of anti-aging and / or anti-inflammatory drugs.

10. Application of R-loop as a target of DHX9 gene in the preparation of anti-aging and / or anti-inflammatory drugs.

11. The application according to claim 10, characterized in that: The drugs include overexpression of DHX9 or silencing of R-loop.

12. The application according to claim 11, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

13. The use according to claim 11, characterized in that The drug is a drug that upregulates DHX9 levels, ATP levels and OCR levels, downregulates R-loop levels, and downregulates the levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β.

Images