Preliminary Study On The Effect Of Regulating Cell Autophagy On The Formation Of Rabbit IPSCs And The Expression Of Related Genes

The technology of induced pluripotent stem cells?iPSCs?-using pluripotent factors provides a new pathway to study the somatic cell reprogramming,which would avoid the ethical problems associated with using human embryos,and brings new views for stem cells research and regenerative medicine.Furthermore,iPS technology is an important tool to study the gene expression regulation pattern,the interaction between proteins and the development of organism.By now,although some achievement has been obtained about the human iPSCs research,the reprogramming mechanism is still unclear.Therefore,study with the animal models is necessary for iPSCs applications on medicine.The morphology of rabbit iPSCs is similar with the human iPSCs,thus,the research of rabbit-iPSCs?RiPSCs?is a hot topic.In this study,four doxycycline?DOX?-induced expression vectors,containing four transcript factors from human Oct4,Sox2,Klf4 and c-Myc,respectively,were delivered into rabbit fibroblasts?REFs?to generate iPSCs by lentivirus infection.The mRNA level of pluripotent genes and the pluripotent of cells were detected.The autophagy was regulated by adding rapamycin in the culture medium,the reprogramming efficiency and gene expression under presence of rapamincy were observed.Laying the foundations for increasing the reprogramming efficiency and improving the quality of RiPSCs.1.Induction of rabbit iPSCs.The four transcript factors?Oct4,Sox2,Klf4 and c-Myc?were delivered into the REFs by lentivirus infection and induced to express via adding DOX.After 2d induced with Dox,the cellular morphology began to change,and the cell clones were observed after a week.The RiPSCs clones had distint characteristics,such as round shape,large nucleolus and scant cytoplasm.The clones were identified by AP straining,the results showed that they were AP straining positive.The RT-PCR results showed that Oct4,Sox2,Klf4 and c-Myc expressed in.the RiPSCs.Immunostaining results demonstrated that the cells expressed the Oct4,Sox2,Stat3 and Ssea1,but no E-cadherin was detected in the clones.In the 150 clones,only 6 strains could be subcultured over 10 passages.2.The effects of cell autophagy regulation on expression of autophagy related genes and the formation of RiPSCs.Firstly,the effects of Rapamycin treatment on expression of autophagy related-genes?ATG?and the formation of RiPSCs were discussed.Rapamycin was added at the first 3 days in the medium,the AP positive clones were counted and the cell morphology after clone-formation was observed.The results showed the optimal concentration of rapamycin was 0.5 nmol·L^-1.qRT-PCR results showed that the expression of ATG5 was stable during induced process.But the expression of ATG7,LC3 and ULK1 were increased during this process?P<0.05?,while the increase level and the stage were different.The mRNA levels of Oct4,Sox2 and Klf4 pluripotent genes were upregulated after rapamycin treatment?P<0.05?.The AP straining showed that the rapamycin treatment would promote the RiPSCs reprogramming efficiency and improve the quality of RiPSCs.In conclusion,RiPSCs could be obtained by using the DOX-inducing lentivirus system,and the induced RiPSCs had pluripotency.Reprogramming efficiency of RiPSCs would be improved by regulating cell autophagy.