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. On the basis that R-loop formed in the cell aging process is eliminated from the source, the adverse effect of genome instability caused by the R-loop is relieved, and RNH1 overexpression virus transfection MSC for cell R-loop elimination is developed. The RNH1 overexpressed MSCs provided by the invention show remarkable anti-aging activity, also have strong adaptive capacity to environment and excellent safety characteristics, can effectively resist harsh in-vivo inflammation and aging microenvironment, avoids tumorigenic risk after cell transplantation, and has good application prospects. The MSCs with stable anti-aging capability can be subsequently applied to remarkably improving the treatment effect of the 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 mesodermal 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), and 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 autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis; sports system diseases such as osteoarthritis; endocrine system diseases such as type II diabetes; circulatory system diseases such as acute myocardial infarction and heart failure; nervous system diseases such as spinal cord injury and brain injury. 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] 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 specifically intervening in mesenchymal stem cell aging cannot be found. For example, current antioxidants (such as vitamin C) or autophagy activators (such as rapamycin) can reverse MSCs aging to some extent in vitro, but the microenvironment in vivo is complex, and there are oxidative stress, chronic inflammation and other microenvironments. After the cells enter the microenvironment in vivo, 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 aging microenvironment in vivo, such as inflammatory factor gradient and cell-cell communication interference. At the same time, the in vitro cell intervention based on drugs has a certain timeliness and cannot achieve stable anti-aging effect. Current genetic 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.

[0004] Therefore, the present application provides a genetic engineering construction method of anti-aging human mesenchymal stem cells and application. SUMMARY

[0005] The present application aims to provide a genetic engineering construction method and application of anti-aging human mesenchymal stem cells, and to solve the technical problem of being difficult to construct stable and effective MSCs with anti-aging ability after in vivo infusion by overexpressing RNH1 to construct stable anti-aging MSCs.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

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

[0008] S1, inserting the target gene RNH1 into a lentivirus system vector to construct a GV492-RNH1 recombinant plasmid, wherein the amino acid sequence of the target gene RNH1 is shown as SEQ ID NO. 1;

[0009] S2, co-transfecting the GV492-RNH1 recombinant plasmid obtained in S1 and a packaging plasmid into a host cell to prepare a lentivirus particle;

[0010] S3, transfecting the lentivirus particle obtained in S2 into human mesenchymal stem cells to obtain genetically engineered RNH1 overexpressing human mesenchymal stem cells for anti-aging or anti-inflammatory.

[0011] Preferably, the construction method of the GV492-RNH1 recombinant plasmid is: obtaining the target gene RNH1 by PCR amplification and cloning using a primer pair; then, the lentivirus system vector and the RNH1 gene sequence are respectively subjected to enzyme cutting using AgeI and NheI restriction endonucleases, and the cloning connection is completed by In-fusion seamless cloning method.

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

[0013] Preferably, the lentivirus system vector is GV492 (pGC-FU-3FLAG-CBh-gcGFP-

[0014] IRES-puromycin).

[0015] Preferably, the host cell is 293T cell, and the packaging plasmid is psPAX2 and pMD2.G.

[0016] Preferably, in step S3, the transfection is specifically: transfecting the lentivirus particle into the human mesenchymal stem cells in a medium containing 1x 10 5In the well of the MSCs, a mixture of RNH1 overexpression lentivirus, transfection auxiliary reagent Polybrene and DMEM complete culture medium mixed in a ratio of 10:86:4 was added, and cultured for 48 hours.

[0017] In one aspect, the application provides the genetically engineered RNH1 overexpression human mesenchymal stem cells prepared by the construction method described above.

[0018] In one aspect, the application provides the use of the RNH1 overexpression human mesenchymal stem cells described above in the preparation of anti-aging and anti-inflammatory drugs.

[0019] Preferably, the drug refers to a drug that achieves anti-aging or anti-inflammatory by up-regulating RNH1 level, down-regulating R-loop level, and down-regulating SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β level, up-regulating ATP level and down-regulating OCR level.

[0020] Preferably, the drug includes RNH1 overexpression or silencing R-loop.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application is based on eliminating R-loop (R loop) formed in the process of cell aging from the source, relieving the adverse effects of genomic instability caused by R-loop, and developing RNH1 overexpression virus transfection MSC aiming at eliminating cell R-loop. It is the first time to propose the strategy of stably overexpressing RNH1 to eliminate genomic R-loop structure, and improve the anti-aging ability of MSCs after infusion in vivo. The MSCs have stable RNH1 overexpression effect and anti-aging function, and are not aimed at a single gene site, but relieve a series of aging phenotypes and genomic instability problems in the aging process by eliminating R-loop. RNH1 overexpression MSCs exhibit significant anti-aging activity, have strong environmental adaptability and excellent safety characteristics, and can effectively resist harsh in vivo inflammatory and aging microenvironment, while avoiding the risk of tumorigenesis after cell transplantation. The MSCs with stable anti-aging ability can be applied to significantly improve the therapeutic effect of MSCs infusion in vivo to treat diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a staining chart and quantitative analysis of different groups of MSCs aging level by β-galactosidase staining. Young refers to young primary MSCs, old refers to old primary MSCs, UV - refers to young passage 3 MSCs without ultraviolet induction of aging, UV +P16 refers to young passage 16 MSCs, P21 refers to young passage 21 MSCs, P3 refers to young passage 3 MSCs induced by UV, P3 is young passage 3 MSCs, P12 refers to naturally aged MSCs of young passage 12);

[0024] Figure 2 is the Western Blot detection of MSCs P16 and P21 protein expression in different groups, and the quantitative analysis of the immunoblotting map;

[0025] Figure 3 is the RT-qPCR detection result of MSCs P16 and P21 mRNA expression level in different groups;

[0026] Figure 4 is the RT-qPCR detection result of MSCs SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β mRNA expression level in different groups;

[0027] Figure 5 is the ELISA detection result of MSCs SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β protein secretion level in different groups;

[0028] Figure 6 is the detection result of ATP level of MSCs in different groups;

[0029] Figure 7 is the detection result of OCR (oxygen consumption rate) level of MSCs in different groups;

[0030] Figure 8 is the fluorescence map and quantitative analysis of S9.6 immunofluorescence detection of R-loop level of MSCs in different groups;

[0031] Figure 9 is the immunoblotting map and quantitative analysis of S9.6 Dotblot detection of R-loop level of MSCs in different groups;

[0032] Figure 10 is the result of transcriptome sequencing RNH1 expression level in MSCs in different groups;

[0033] Figure 11 is the flow chart of OE-RNH1-MSCs (RNH1 overexpression MSCs) infusion treatment of CIA arthritis mouse model;

[0034] Figure 12 is the Western Blot detection of MSCs RNH1 protein immunoblotting map and quantitative analysis result (Vector refers to empty load, OE-RNH1 refers to overexpression RNH1 virus vector);

[0035] Figure 13 are the fluorescence images and quantitative analysis of S9.6 immunofluorescence detection of R-loop levels of MSCs overexpressing RNH1 in different groups (old refers to old primary MSCs, old + OE-RNH1 refers to old primary MSCs overexpressing RNH1, UV refers to UV-induced senescent passage 3 MSCs, UV + OE-RNH1 refers to UV-induced senescent passage 3 MSCs overexpressing RNH1, P12 refers to young naturally senescent MSCs passaged to passage 12, and P12 + OE-RNH1 refers to young naturally senescent MSCs passaged to passage 12 overexpressing RNH1); + are the fluorescence images and quantitative analysis of S9.6 immunofluorescence detection of R-loop levels of MSCs overexpressing RNH1 in different groups (old refers to old primary MSCs, old + OE-RNH1 refers to old primary MSCs overexpressing RNH1, UV refers to UV-induced senescent passage 3 MSCs, UV + OE-RNH1 refers to UV-induced senescent passage 3 MSCs overexpressing RNH1, P12 refers to young naturally senescent MSCs passaged to passage 12, and P12 + OE-RNH1 refers to young naturally senescent MSCs passaged to passage 12 overexpressing RNH1);

[0036] Figure 14 are the immunoblot images and quantitative analysis of S9.6 Dotblot detection of R-loop levels of MSCs overexpressing RNH1 in different groups;

[0037] Figure 15 are the staining images and quantitative analysis of β-galactosidase staining detection of senescence levels of MSCs overexpressing RNH1 in different groups;

[0038] Figure 16 are the results of RT-qPCR detection of mRNA expression levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6, and TGF-β of MSCs overexpressing RNH1 in different groups;

[0039] Figure 17 are the results of ELISA detection of protein secretion levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6, and TGF-β of MSCs overexpressing RNH1 in different groups;

[0040] Figure 18 are the results of ATP level detection of MSCs overexpressing RNH1 in different groups;

[0041] Figure 19 are the results of OCR (oxygen consumption rate) level detection of MSCs overexpressing RNH1 in different groups;

[0042] Figure 20 are the results of local gross images, tissue HE staining, and safranin-fast green staining of mouse ankle joints after treatment with OE-RNH1-MSCs;

[0043] Figure 21 are the results of arthritis scores and palm thickness measurements of mice after treatment with OE-RNH1-MSCs. DETAILED DESCRIPTION

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0045] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0046] 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.

[0047] Example 1 Extraction of MSCs to construct a senescent cell model

[0048] 1. Experimental methods

[0049] 1) Construction of a senescent cell model

[0050] Construction of a primary senescent cell model: 30 ml of bone marrow was extracted from an elderly healthy volunteer and a young healthy volunteer via the posterior superior iliac spine, respectively, and was heparin-anticoagulated. 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 the 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. The MSCs were subcultured to the 3rd generation, inoculated in a 6-well plate at a density of 1 x 10 5 / well, to obtain elderly subcultured MSCs and young subcultured MSCs.

[0051] Construction of a subcultured senescent cell model: the young MSCs were subcultured to the 12th generation and inoculated in a 6-well plate at a density of 1 x 10 5 / well, to obtain young subcultured naturally senescent MSCs.

[0052] Construction of an ultraviolet-induced senescent cell model: the young primary MSCs were subcultured to the 3rd generation and inoculated in a 6-well plate at a density of 1 x 10 5 / well. After a single irradiation, the MSCs were cultured for 72 hours to obtain ultraviolet-induced senescent MSCs. 2

[0053] 2) Determination of the indicators in the MSCs of each senescent cell model constructed in 1)

[0054] The β-galactosidase staining kit was used to detect the senescence level of the MSCs, Western blot was used to detect the P16 / P21 protein expression of the MSCs, and the RT-qPCR instrument was used to detect the P16 / P21 mRNA expression of the MSCs. ​

[0055] RT-qPCR was used to detect the expression of SASP mRNA in MSCs, ELISA kit was used to detect the secretion of SASP in MSCs; ATP detection kit was used to detect the ATP level of MSCs, OCR detection kit was used to detect the OCR (oxygen consumption rate) level of MSCs; immunofluorescence staining and Dotblot were used to detect the R-loop level in MSCs.

[0056] 2. Experimental results

[0057] 2.1 β-galactosidase staining of senescent and young MSCs

[0058] The results of β-galactosidase staining showed that, compared with young group MSCs, the β-galactosidase staining of old group MSCs was darker, and the proportion of β-galactosidase staining positive cells was more; compared with UV- group MSCs, the β-galactosidase staining of UV+ group MSCs was darker, and the proportion of β-galactosidase staining positive cells was more; compared with P3 group MSCs, the β-galactosidase staining of P12 group MSCs was darker, and the proportion of β-galactosidase staining positive cells was more (see Figure 1 ).

[0059] 2.2 Western Blot and RT-qPCR detection of P16 / P21 expression in old and young MSCs

[0060] The results of Western Blot and RT-qPCR detection showed that, compared with young group MSCs, the expression of P16 and P21 protein and mRNA in old group MSCs was higher; compared with UV- group MSCs, the expression of P16 and P21 protein and mRNA in UV+ group MSCs was higher; compared with P3 group MSCs, the expression of P16 and P21 protein and mRNA in P12 group MSCs was higher (see Figure 2 and Figure 3 ).

[0061] 2.3 RT-qPCR and LISA detection of SASP expression and secretion in old and young MSCs

[0062] The expression of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β mRNA and protein secretion level of the old group MSCs were higher than those of the young group MSCs; the expression of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β mRNA and protein secretion level of the UV+ group MSCs were higher than those of the UV- group MSCs; the expression of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β mRNA and protein secretion level of the P12 group MSCs were higher than those of the P3 group MSCs Figure 4 and Figure 5 ).

[0063] 2.4 Detection of ATP level of old and young MSCs

[0064] The ATP level of the old group MSCs was lower than that of the young group MSCs; the ATP level of the UV+ group MSCs was lower than that of the UV- group MSCs; the ATP level of the P12 group MSCs was lower than that of the P3 group MSCs (see Figure 6 ).

[0065] 2.5 Detection of OCR level of old and young MSCs

[0066] The OCR level of the old group MSCs was lower than that of the young group MSCs; the OCR level of the UV+ group MSCs was lower than that of the UV- group MSCs; the OCR level of the P12 group MSCs was lower than that of the P3 group MSCs (see Figure 7 ).

[0067] 2.6 Detection of R-loop level of old and young MSCs by S9.6 immunofluorescence and S9.6 Dotblot

[0068] The results showed that the R-loop level of the old group MSCs was higher than that of the young group MSCs; the R-loop level of the UV+ group MSCs was higher than that of the UV- group MSCs; the R-loop level of the P12 group MSCs was higher than that of the P3 group MSCs (see Figure 8 and Figure 9 ).

[0069] In summary, the R-loop level of the senescent MSCs was increased, the β-galactosidase staining was quantified, the P16 / P21 protein and mRNA level was high, the SASP expression and secretion level was high, and the ATP and OCR levels were low.

[0070] Example 2 Construction and application of RNH1 overexpression lentivirus

[0071] The present embodiment is found by transcriptome sequencing data that the TPM value of RNH1 of MSCs in the young group is significantly reduced, which indicates that RNA helicase protein RNH1 is significantly down-regulated in mesenchymal stem cells of the elderly Figure 10 That is, RNH1 down-regulation mediates genome R-loop accumulation, so RNH1 overexpression lentivirus is constructed to verify the effect of overexpression of RNH1 on mesenchymal stem cells resisting aging, and RNH1 overexpression MSCs with stable anti-aging ability are used for infusion treatment of CIA arthritis model mice, and good treatment effect is obtained.

[0072] I. Construction of RNH1 overexpression lentivirus

[0073] (1) Plasmid construction:

[0074] The human RNH1 gene (NM_001357) is obtained from the cDNA library of Genechem (Shanghai Jikai Gene Medical Technology Co., Ltd.), and the following primers are used: RNH1 forward primer: 5'-ATGAAAGCGCAGAGCCGTAT-3', RNH1 reverse primer: 5'-CTCCCATGTAGGAAAA CGTGTC-3'; the RNH1 gene with the amino acid sequence as shown in SEQ ID NO. 1 is cloned.

[0075] The lentivirus vector plasmid GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin) (purchased from Shanghai Jikai Gene Medical Technology Co., Ltd.) and RNH1 gene sequence are respectively digested with AgeI and NheI restriction endonuclease, and the cloning connection is completed by In-fusion seamless cloning (recombination) method. The recombinant vector is identified by DNA sequencing detection.

[0076] The amino acid sequence of RNH1 (SEQ ID NO. 1) is:

[0077] MSWLLFLAHRVALAALPCRRGSRGFGMFYAVRRGRKTGVFLTWNECRAQVDRFPAARFKKFATEDEAWAFVRKSASPEVSEGHENQHGQESEAKASKRLREPLDGDGHESAEPYAKHMKPSVEPAPPVSRDTFSYMGDFVVVYTDGCCSSNGRRRPRAGIGVYWGPGHPLNVGIRLPGRQTNQRAEIHAACKAIEQAKTQNINKLVLYTDSMFTINGITNWVQGWKKNGWKTSAGKEVINKEDFVALERLTQGMDIQWMHVPGHSGFIGNEEADRLAREGAKQSED.

[0078] (2) Lentivirus production:

[0079] The above-constructed viral vector and two auxiliary packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells using Lipofectamine 2000 (purchased from Invitrogen; Thermo Fisher Scientific, Inc.). The culture supernatant containing the virus was collected 72 hours after transfection, and the cell debris was removed by rapid centrifugation at 1000xg, and then sterilized by 0.45μm cellulose acetate filter membrane.

[0080] The virus titer was determined by flow cytometry (FACS) analysis of the proportion of GFP-positive 293T cells, about 1x10 9 Transduction units (TU) per mL of culture medium.

[0081] II. Application of RNH1 overexpression lentivirus

[0082] 1. Experimental method

[0083] (1) In vitro cell experiment

[0084] 1) The RNH1 overexpression lentivirus was used to transfect the young and old primary MSCs prepared in Example 1, passaged induced senile MSCs, and ultraviolet induced senile MSCs for 48 hours, as follows: 1x 10 5 After the MSCs were plated in a 12-well plate, the supernatant was removed, and the RNH1 overexpression lentivirus, transfection auxiliary reagent, and complete culture medium were mixed in a ratio of 10:86:4, and then 2ml / well was added and cultured for 48 hours.

[0085] 2) Western blot was used to detect the RNH1 protein expression of MSCs, RT-qPCR was used to detect the RNH1 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.

[0086] (2) In vivo animal experiments

[0087] 1) Construct CIA arthritic mouse model, the specific construction scheme is as follows:

[0088] On day 1, DBA / 1 male mice (6-8 weeks old, body weight 20-30 grams) were subcutaneously injected with 100 microliters (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). On day 21, a booster immunization was performed using 100 microliters (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.

[0089] Arthritis severity assessment:

[0090] 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 range redness or swelling; 4 points: joint ankylosis or severe deformity. The total arthritis score of each mouse was the sum of the scores of the four legs of the mouse.

[0091] Hind paw thickness measurement: The thickness of the mouse hind paw was measured using a vernier caliper to assess the severity of inflammation.

[0092] 2) OE-RNH1-MSCs infusion treatment of CIA arthritic mouse model

[0093] The specific process is shown in Figure 11, construct the mice ankle joint local gross image, tissue HE staining, safranin O fast green staining, and detect the arthritis score and measure the palm thickness after the construction is successful.

[0094] 2. Experimental results

[0095] (1) In vitro cell experiment

[0096] Western Blot shows that the RNH1 protein level is overexpressed in the OE-RNH1 group: relative to the MSCs of the Vector group, the immunoblotting and quantification of the MSCs of the OE-RNH1 group shows that the RNH1 protein expression is significantly increased (see Figure 12 ).

[0097] S9.6 immunofluorescence and Dotblot show that the R-loop level of MSCs overexpressing RNH1 is significantly reduced: relative to the MSCs of the old group, the MSCs of the old + OE-RNH1 group have a lower R-loop level; relative to the MSCs of the UV+ group, the MSCs of the UV+OE-RNH1 group have a lower R-loop level; relative to the MSCs of the P12 group, the MSCs of the P12+OE-RNH1 group have a lower R-loop level (see Figure 13 and 14 ).

[0098] β-galactosidase staining shows that the senescence level of MSCs overexpressing RNH1 is significantly reduced: relative to the MSCs of the old group, the MSCs of the old + OE-RNH1 group are more lightly stained and have a lower proportion of positively stained cells; relative to the MSCs of the UV+ group, the MSCs of the UV+OE-RNH1 group are more lightly stained and have a lower proportion of positively stained cells; relative to the MSCs of the P12 group, the MSCs of the P12+OE-RNH1 group are more lightly stained and have a lower proportion of positively stained cells (see Figure 15 ).

[0099] RT-qPCR and ELISA show that the expression and secretion levels of SASP of MSCs overexpressing RNH1 are significantly reduced: relative to the MSCs of the old group, the MSCs of the old + OE-RNH1 group have lower mRNA expression and protein secretion levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β; relative to the MSCs of the UV+ group, the MSCs of the UV+OE-RNH1 group have lower mRNA expression and protein secretion levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β; relative to the MSCs of the P12 group, the MSCs of the P12+OE-RNH1 group have lower mRNA expression and protein secretion levels of SASP molecules IL-1A, IL1B, CCL2, CCL5, IL-6 and TGF-β (see Figure 16 and 17 ).

[0100] ATP detection ATP levels of MSCs overexpressing RNH1 were significantly increased: the ATP levels of MSCs in the old + OE-RNH1 group were higher than those in the old group; the ATP levels of MSCs in the UV+OE-RNH1 group were higher than those in the UV+ group; the ATP levels of MSCs in the P12+OE-RNH1 group were higher than those in the P12 group (see Figure 18 ).

[0101] OCR detection OCR levels of MSCs overexpressing RNH1 were significantly increased: the OCR levels of MSCs in the old + OE-RNH1 group were higher than those in the old group; the OCR levels of MSCs in the UV+OE-RNH1 group were higher than those in the UV+ group; the OCR levels of MSCs in the P12+OE-RNH1 group were higher than those in the P12 group (see Figure 19 ).

[0102] In summary, after overexpression of RNH1, the mRNA and protein levels of MSCs RNH1 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, which proved that overexpression of RNH1 could significantly promote the ability of MSCs to resist aging.

[0103] (2) In vivo animal experiments

[0104] OE-RNH1-MSCs infusion treatment of CIA arthritis mouse model, ankle local gross image, tissue HE staining, ponceau solid green staining: relative to CIA group and CIA+MSCs infusion treatment group mice, CIA+OE-RNH1 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 (see Figure 20 ).

[0105] OE-RNH1-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-RNH1 MSCs infusion treatment group mice arthritis score is lower, palm thickness is thinner (see Figure 21 ).

[0106] In summary, OE-RNH1-MSCs infusion treatment of CIA mouse arthritis model, joint local inflammation is relieved, arthritis score and palm thickness are reduced.

[0107] In summary, the present application uses lentivirus to stably overexpress RNH1 as a strategy, and clears R-loop in genome as a target to develop anti-aging human mesenchymal stem cells. The strategy does not take a certain gene as a target, but aims at the core mechanism of cell aging, i.e., genome instability, and constructs anti-aging MSCs by overexpressing RNH1 to clear the important 'perpetrator' of genome instability, i.e., R-loop structure. Stable overexpression of RNH1-MSCs is expected to stably clear R-loop structure in cells, better cope with various stimuli inducing cell aging when facing complex in-vivo microenvironment after in-vivo infusion, and thus stably play a therapeutic role and improve MSCs in-vivo infusion treatment scheme.

[0108] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A method for constructing genetically engineered human mesenchymal stem cells overexpressing RNH1, characterized in that, Includes the following steps: 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. 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 genetically engineered human mesenchymal stem cells that overexpress RNH1 for anti-aging or anti-inflammatory purposes.

2. The construction method according to claim 1, 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.

3. The construction method according to claim 2, characterized in that, The primer pair includes a forward primer: 5'-ATGAAAGCGCAGAGCCGTAT-3' and a reverse primer: 5'-CTCCCATGTAGGAAA ACGTGTC-3'.

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

5. The construction method according to claim 1, characterized in that, The host cell is a 293T cell, and the packaging plasmids are psPAX2 and pMD2.G.

6. The construction method according to claim 1, characterized in that, In step 3, the transfection specifically involves: in a solution containing 1x10 5 Add a mixture of RNH1 overexpression lentivirus, transfection aids and complete culture medium in a ratio of 10:86:4 to each well of MSCs and incubate for 48 hours.

7. Human mesenchymal stem cells overexpressing RNH1 prepared by the construction method according to any one of claims 1-6.

8. The use of human mesenchymal stem cells overexpressing RNH1 as described in claim 7 in the preparation of anti-aging and anti-inflammatory drugs.

9. The application according to claim 8, characterized in that, 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.

10. The application according to claim 10, characterized in that, The drugs include those that overexpress RNH1 or silence the R-loop.

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