Rapamycin Induces Autophagy And Delays Aging In Bovine Fibroblasts

Cell aging is an inevitable phenomenon in the process of cell culture.Delaying the aging process of cells and improving the vitality of cell passage play an important role in obtaining immortalized cells,stem cell passage and screening transgenic positive cells.Autophagy is one of the mechanisms of intracellular self-repair,maintaining the balance of cell metabolism and participating in the process of cell growth and development such as cell aging.With the deepening of studies on autophagy,people find more and more evidence that autophagy plays an important role in delaying cell aging.The autophagy level of cells is closely related to the aging process.In the aging process,the autophagy level decreases,and the decrease of autophagy level can accelerate the aging process.Meanwhile,the increase of autophagy level can delay the aging process.Improving the level of autophagy and delaying cell aging has become a hot scientific issue,attracting the attention of scientists from all over the world,which is of great significance for the protection of cell genetic resources and the improvement of diseases related to aging.Rapamycin is a novel immunosuppressant for organ transplantation.Rapamycin induces and promotes autophagy by inhibiting mTOR.In this study,rapamycin was used to treat adult bovine skin fibroblasts,aiming to improve the aging phenomenon of adult fibroblasts and prolong cell life by promoting autophagy,so as to protect its genetic resources and provide ideal biological materials for nuclear transplantation.In this study,adult bovine fibroblasts aged to passage 13 were treated with rapamycin(RAPA).The expression of CDKN2 A,CDKN1A and β-galactosidase were detected and the changes of morphology of autophagy in the cells were observed.The expression of green dot-like aggregated GFP-LC3 fusion protein was observed by transfection of pEX-GFP-hLC3 WT plasmid,and autophagy-associated protein p-mTOR,mTOR,LC3B-II/ LC3B-I was detected by Western Blot.The experimental results of cell aging model showed that compared with the primary cells,the 13 th generation cells grew slowly,the cell proliferation ability was slow,and the β-galactosidase positive staining rate in the cells was significantly increased(P<0.05),CDKN2 A and CDKN1 A mRNA levels and protein expression were also significantly increased(P < 0.05).The results of these indicators fully indicate that P13 cells show significant aging compared with primary cells,and the cell aging model is successfully established..The experimental results of rapamycin treatment aging model showed that the age d aging adult bovine fibroblasts treated with RAPA significantly reduced the β-galacto sidase staining rate of aged cells.and reduced the expression levels of CDKN2 A,CDK N1 A mRNA and protein related to cell senescence(P<0.05).The results of these indi cators indicated that rapamycin treatment could improve the aging status of aging cell s.From the primary passage to the 13 th generation of fibroblasts,the number of intracellular autophagosomes decreased,the expression of autophagy specific protein LC3 was also weakened,the phosphorylation of mTOR protein was enhanced,and the inhibition effect was reduced,and the level of intracellular autophagy was gradually reduced.The results of autophagy level test after rapamycin showed that autophagosomes increased in aging cells,the number of GFP-LC3 green fluorescent spots increased,mTOR expression was inhibited,and the ratio of autophagy related protein LC3-II/LC3-I increased,indicating that RAPA treatment of aging bovine fibroblasts increased autophagy level.The conclusion of this study is that rapamycin can delay cell aging by increasing autophagy level of bovine fibroblasts.