CN-121015703-B - Genetic engineering construction method and application of anti-aging human mesenchymal stem cells

Abstract

The invention belongs to the technical field of genetic engineering and cell transformation, and discloses a genetic engineering construction method and application of an anti-aging human mesenchymal stem cell. The invention develops MSC transfected by RNH1 over-expression virus for eliminating cell R-loop based on eliminating the R-loop formed in the cell aging process from the source and relieving the adverse effect of genome instability caused by the R-loop. The RNH1 over-expressed MSCs provided by the invention show remarkable anti-aging activity, have strong environment adaptability and excellent safety characteristics, can effectively resist severe in-vivo inflammation and aging microenvironment, avoid the tumorigenic risk after cell transplantation, and can be applied to remarkably improve the curative effect of MSCs infusion in vivo for treating diseases.

Inventors

• LI JINTENG
• LIU ZHIDONG
• Ye Guiwen
• Lan Zhexiao
• XU PEITAO
• XU XIAOJUN
• CHEN CHAOQIANG

Assignees

• 中山大学附属第八医院（深圳福田）

Dates

Publication Date

20260508

Application Date

20250915

Claims (8)

1. 1. The application of the anti-aging human mesenchymal stem cells in preparing the anti-arthritis medicament is that the anti-aging human mesenchymal stem cells are derived from bone marrow and are the human mesenchymal stem cells which over-express RNH1, and the amino acid sequence of the RNH1 is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the method for constructing anti-aging human mesenchymal stem cells comprises the steps of: S1, inserting a target gene RNH1 into a lentiviral system vector to construct a GV492-RNH1 recombinant plasmid; s2, co-transfecting the GV492-RNH1 recombinant plasmid obtained in the S1 and the packaging plasmid into a host cell to prepare slow virus particles; s3, transfecting the slow virus particles obtained in the step S2 into human mesenchymal stem cells to obtain the anti-aging human mesenchymal stem cells.
3. 3. The application of the recombinant GV492-RNH1 plasmid according to claim 2, wherein the construction method comprises the steps of cloning the target gene RNH1 after PCR amplification by using a primer pair, and then carrying out enzyme digestion on a lentiviral system vector and an RNH1 gene sequence by using AgeI and NheI restriction enzymes respectively, and completing cloning connection by an In-fusion seamless cloning method.
4. 4. The use according to claim 3, wherein the primer pair comprises a forward primer 5'-ATGAAAGCGCAGAGCCGTAT-3' and a reverse primer 5'-CTCCCATGTAGGAAAACGTGTC-3'.
5. 5. The use according to claim 3, wherein the lentiviral system vector is pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin.
6. 6. The use according to claim 2, wherein the host cell is a 293T cell.
7. 7. The use according to claim 2, wherein the packaging plasmids are psPAX and pmd2.G.
8. 8. The use according to claim 2, wherein in step S3, the transfection is performed by adding a mixture of lentiviral particles, transfection aid and complete medium obtained in step S2, mixed thoroughly in a ratio of 10:86:4, to a well plate containing 1X 10 5 MSCs, and incubating for 48 hours.

Description

Genetic engineering construction method and application of anti-aging human mesenchymal stem cells Technical Field The invention relates to the technical field of genetic engineering and cell transformation, in particular to a construction method of mesenchymal stem cells, and in particular relates to a genetic engineering construction method and application of anti-aging human mesenchymal stem cells. Background Mesenchymal stem cells (MSCs, MESENCHYMAL STEM CELLS) belong to pluripotent adult stem cells derived from mesoderm, exist in tissues such as bone marrow, fat, umbilical cord, placenta, dental pulp, synovium and the like, can be expanded and differentiated into various cell types such as bone, cartilage, fat, nerve, cardiac muscle, liver cells and the like in vitro for a long time, 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 during allograft, rarely cause immune rejection, and can regulate microenvironment and promote tissue regeneration through the inflammatory and homing effect and secretion effect. The mesenchymal stem cell infusion treatment has obvious potential in the treatment of various diseases, and has been used for autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, motor system diseases such as osteoarthritis, endocrine system diseases such as type II diabetes, circulatory system diseases such as acute myocardial infarction and heart failure, and nervous system diseases such as spinal cord injury and brain injury. Aging of MSCs refers to the biological process by which under physiological or pathological conditions, self-renewal capacity, multipotent differentiation potential and paracrine function gradually decline due to cumulative injury. This process is accompanied by characteristic changes in cell morphology, gene expression and metabolic activity, and finally manifests as a loss of repair and regeneration capacity, which is one of the important mechanisms of the whole body of aging and the occurrence of aging-related diseases. Although research on mesenchymal stem cell aging has been well progressed in recent years, the mechanism of cell aging is quite complex, the exact cause of cell aging cannot be ascertained at present, and a method for targeted intervention of mesenchymal stem cell aging cannot be found. Although the existing 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, and microenvironments such as oxidative stress, chronic inflammation and the like exist, and after cells enter the microenvironment in vivo, the cells can be changed due to the influence of the microenvironment, so that the effect is obviously reduced. Furthermore, in vitro culture conditions cannot mimic the dynamic changes of the aging microenvironment in vivo, such as inflammatory factor gradients, intercellular communication interference. Meanwhile, in-vitro cell intervention based on medicines has certain timeliness, and a stable anti-aging effect cannot be achieved. The current genetic engineering usually aims at over-expression or knockout of a single gene locus, but the multi-gene interaction network in the cell aging process is complex, and the whole cell function is difficult to change by single editing. Therefore, the invention provides a genetic engineering construction method and application of an anti-aging human mesenchymal stem cell. Disclosure of Invention The invention aims to provide a genetic engineering construction method and application of an anti-aging human mesenchymal stem cell, and the technical problem that stable and effective MSCs with anti-aging capacity after in-vivo infusion are difficult to construct in the prior art is solved by constructing stable anti-aging MSCs through over-expression RNH 1. In order to achieve the above purpose, the present invention adopts the following technical scheme: in one aspect, the invention provides a method for constructing genetically engineered human mesenchymal stem cells over-expressing RNH1, comprising the following steps: S1, inserting a target gene RNH1 into a lentiviral 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; s2, co-transfecting the GV492-RNH1 recombinant plasmid obtained in the S1 and the packaging plasmid into a host cell to prepare slow virus particles; S3, transfecting the slow virus particles obtained in the S2 into human mesenchymal stem cells to obtain the anti-aging or anti-inflammatory genetically engineered human mesenchymal stem cells over-expressing RNH 1. Preferably, the construction method of the GV492-RNH1 recombinant plasmid comprises the steps of performing PCR amplification by using a primer pair, cloning to obtain a target gene RNH1, and performing enzyme digestio