Regulation And Mechanism Of HMGA2 On Senescence Of Mesenchymal Stem Cells From Aged Rats Bone Marrow

Objective:The recombinant plasmid pCDH-CMV-HMGA2-EF1-copGFP was constructed by using the lentiviral expression vector p CDH-CMV-MCS-EF1-copGFP to make HMGA2 highly expressed in BMSCs of aged rats.This study observed and investigated the effect of HMGA2 on the senescence of BMSCs in aged rats and its possible mechanism.Methods:SD rats were selected at the age of 2 weeks and 20 months.Bone marrow mesenchymal stem cells（BMSCs）were isolated from rat tibia and femur bone and purified using adherent methods,and cultured to passage 3.Flow cytometry was used to determine the surface marker CD11b/c,CD45 and CD90.β-gal staining was used to detect the senescence of BMSCs,as well as q PCR to measure the expression level of HMGA2 in the cells.The expression vector of HMGA2 lentiviral vector was further constructed.The cultured rat SD rat bone marrow mesenchymal stem cells were divided into two experimental groups,which were infected with Empty-Lenti and HMGA2-Lenti virus,respectively.1.The expression of HMGA2 gene in Empty-Lenti and HMGA2-Lenti BMSCs was detected by real-time fluorescence quantitative PCR,and the expression of HMGA2 was analyzed by 2^{（-△△ct）}method;2.The number of senescent cells in Empty-Lenti and HMGA2-Lenti cells was detected byβ-galactosidase（β-gal）staining;3.The content of NO in supernatant of Empty-Lenti and HMGA2-Lenti cells was detected by nitric oxide（NO）detection kit;4.The number of clones formed by two groups of BMSCs was compared by clone formation unit（CFU-F）analysis;5.The expression level of p53and p21 gene in Empty-Lenti and HMGA2-Lenti cells was detected by real-time fluorescence quantitative PCR.After myocardial infarction（MI）model was induced,DiI stained BMSCs with null or HMGA2 plasmid were injected around the MI site.Rats were further sacrificed 3 days after surgery and hearts were collected to determine the survival rate of injected BMSCs.Results:By using bone plastic adherent P3 generation BMSCs,the cell morphology showed whirlpool growth.Flow cytometry showed the surface markers of the putative BMSCs were CD90 positive,CD11b/c negative and CD45 negative.The results showed different senescent level between cells isolated from young and aged rats,and showed the negative relation between the expression level of HMGA2 and senescence.After successfully constructing the lentiviral plasmid of HMGA2 and efficiently infected SD rats in the senescence BMSCs,qPCR detection showed that the expression of HMGA2 in the HMGA2-Lenti group was significantly higher than that in the Empty-Lenti group.Nitric oxide test showed that the content of nitric oxide in the supernatant of HMGA2-Lenti group was significantly lower than that in Empty-Lenti group.Beta galactosidase staining was found senescent cells was significantly reduced in HMGA2-Lenti group than in Empty-Lenti group.p53 and p21 were detected with q PCR,the gene expression level in HMGA2-Lenti group decreased significantly than in Empty-Lenti group.After the injection of modified BMSCs in myocardial infarction site,the survival rate of HMGA2-Lenti group was higher than that of group Empty-Lenti.Conclusion:The expression of HMGA2 in rat BMSCs was significantly decreased with the increase of age.In vitro experiments showed that high expression of HMGA2could improve the anti-aging and cell cloning ability of BMSCs in aged rats.In vivo experiments showed that high expression of HMGA2 could promote senile survival rate of BMSCs after transplantation in peripheral regions of myocardial infarction.