Effects Of Autophagy And Energy Metabolism On Induction Efficiency Of Rabbit Ipscs

Induced puripotent Stem cells(iPSCs)contain numerous changes in cellular physiological status,including increased nuclear-to-plasma ratio,mitochondrial reduction,and autophagy et al..Our previous study found that autophagy is closely related to the process of rabbit somatic cell reprogramming to induce pluripotent stem cells(RiPSCs),but the mechanism is still unclear.In this study,we investigated the changes of autophagy and energy metabolism during RiPSCs induction,and then studied the effects of Rapamycin(Rapa)on the induction of RiPSCs.It lays a foundation for understanding the role of autophagy in reprogramming and enriching the reprogramming mechanism.The results are as follows:1 Changes in autophagy during induction of rabbit iPSCs:OCT4,SOX2,C-MYC,KLF4(OSKM)were introduced into rabbit fibroblasts(REF)by tetracycline(Dox)-induced lentiviral vector,and the reprogramming process was observed.Cell morphology was changed,and the obtained RiPSCs were analyzed by immunofluorescence and RT-PCR pluripotency.RT-PCR and transmission electron microscopy were used to analyze the changes of autophagy during reprogramming.The results showed that during reprogramming,cells became smaller,nuclei became larger,cell edge refraction was strong,and cells with morphological changes were clearly distinguished from untransformed cells,and cell colonies were formed.Immunofluorescence results showed that the RiPSCs clone expressed OCT4,SSEA4,and NANOG.During reprogramming,the expression levels of pluripotency genes C-MYC,KLF4,SOX2 and OCT4 increased first and then decreased.The expression of autophagy-related genes also increased first and then decreased.The results of transmission electron microscopy showed that the autophagosome-like structure also showed a trend of increasing first and then decreasing during reprogramming.The above results indicated that the expression of pluripotency genes and the level of autophagy in the reprogramming process of RiPSCs increased first and then decreased.2 Metabolic changes in rabbit iPSCs induction process:This experiment used Mitotracker mitochondria-specific labeling and RT-PCR to detect the mRNA relative expression of metabolic related genes;meanwhile,using the Seahorsse XF24 Extracellular Analyzer(XF)pair The cells in the reprogramming process are tested for mitochondrial respiration and glycolysis,and further analyze the cellular energy metabolism phenotype to assess the type of metabolism in which the cells are located.The results showed that the mitochondrial morphology changed from long rod to small dot during reprogramming,and the number decreased rapidly.The expression levels of mitochondria-related genes NRF1 and PCG1? increased first and then decreased during reprogramming.The expression levels of TFAM and Rad51 decreased first and then increased during reprogramming.The expression levels of glycolytic-related genes PFK and HK2 were highest in RiPSCs,and the expression level of PK in RiPSCs was the lowest.XF test results showed that REF,induced 5d cells,RiPSCs metabolic capacity gradually increased;REF metabolism was relatively weak,biased to resting state;RiPSCs metabolism is strong,belonging to high energy demand type cells,more likely to occur glycolysis.The above results indicated that the mitochondrial morphology of cells changed from long rod to small dot and the number decreased rapidly during reprogramming;REF metabolic capacity was relatively weak and biased to rest;RiPSCs were proliferating and more prone to glycolysis..3 Effect of promoting autophagy on induction of RiPSCs:Based on the previous experimental research,add Rapa to promote autophagy,explore the regulation of autophagy on the reprogramming efficiency of RiPSCs,pluripotency-related genes during reprogramming,autophagy-related genes expression and effects of cellular metabolism.The results showed that the addition of 0.5nM Rapa increased autophagy significantly increased the number of alkaline phosphatase-positive clones and the expression of pluripotency genes C-MYC and OCT4 in RiPSCs;the expression level of autophagy-related genes increased during reprogramming.The cell autophagosome-like structure is increased.XF results showed that Rapa inhibited cell mitochondrial respiratory metabolism during reprogramming and reduced cell metabolism of glycolysis.The above results indicate that promoting autophagy reduces the bioenergy of the cells and improves the efficiency of reprogramming,but also weakens the ability of cells to resist stress from the outside.In conclusion,during the induction of RiPSCs,the level of autophagy increased first and then decreased,and the metabolic capacity of cells increased gradually.The addition of rapamycin promoted the level of autophagy canimprove reprogramming efficiency by reducing the bioenergy of cells,but at the same time,it also weakens the ability of cells to resist stress from the outside world.