| Objective: To investigate the effects of adenovirus-infected mouse embryonic fibroblasts(MEFc)carrying transcription factors(Oct4,Sox2,Klf4,c-Myc),to induce pluripotent stem cells(iPSC),and to analyze the effects of reproducibility p53 gene,MDM2 gene and Aurka gene,which provided the idea of ??increasing the induction efficiency of pluripotent stem cells.Methods: 1.The MEFc was resuscitated.After MEFc growth was good,adenovirus with 4 transcription factors(OSCK)was added and repeated infection was observed twice.The morphological changes of the cells were observed.(IFN-PAGE)staining and stem cell marker gene detection;The transfected cells were cultured according to the morphological observation,tumorigenicity,alkaline phosphatase.After induction,1week after induction and 2 weeks after induction,the transcription of p53 gene,MDM2 gene and Aurka gene were measured by Real-Time PCR technique.The data were analyzed by SPSS 17.0 software.The data of each time were analyzed by one-way ANOVA.The statistical test was P<0.05.Results: 1.The pluripotency of the induction of cells: a,morphological changes: the cell boundary can be observed clear,cell body smaller mouse fibroblasts become larger,rounded,shorter length,a few were variability,There is still a significant protrusion,but the protrusion length becomes shorter.B,tumor formation observation: about 7-8 weeks after the rat root can be seen obvious 2-3 teratoma,hand touch medium mass hardness,can be sliding,and the surrounding tissue without adhesion,smooth surface.Remove the mass of the mass,you can see the block pale red block oval column,the size of about 1cm × 0.5cm × 0.3cm.We will be removed from the tissue section slices,HE staining treatment,showing the nucleus into blue,cytoplasm into pink,can be seen in a large number of goblet cells composed of glandular tissue,blood vessels,a lot of adipose tissue.C,alkaline phosphatase identification: showing a large number of alkaline phosphatase positive particles.D,stem cell marker gene detection: Oct4,Sox2 and Nanog in the induced cells were expressed.There were significant differences in the expression of p53 during induction(P <0.05).The expression of p53 in the induction process was significantly higher than that in the control group(P <0.05).2,after transfection and 1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P<0.05).3,1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P <0.05).The transcription of p53 was elevated after transfection of the four-factor adenovirus,which was higher after transfection for 1 week and decreased after 2 weeks of transfection.The changes of MDM2 in the process of induction were: 1,before and after transfection,1 week after transfection,2 weeks after transfection,RQ was significantly different(P <0.05).2,after transfection and 1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P<0.05).3,1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P <0.05).MDM2 transcription in the four factors after adenovirus transfection decreased,1week decreased significantly,but after 2 weeks of transfection appeared to rise.And p53 gene changes inversely proportional to the law.(P <0.05).Compared with the two weeks after transfection,the difference of RQ was not significant(P <0.05).There was no significant difference between the two groups(P> 0.05).Significant(P> 0.05).2,after transfection and 1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P<0.05).3,1 week after transfection and two weeks after transfection,the difference was significant and statistically significant(P <0.05).It was thought that the transcription of Aurka was significantly increased after transfection of the four factor adenovirus,which was higher at 1 week than that after transfection,but at 2 weeks after transfection and the normal level of Aurka was significantly higher than that of p53 gene.The expression of p53 protein was significantly increased by using MDM2 and Aurka inhibitors.Conclusions :1.The process of reprogramming makes the increment of p53 levels in the i PS cells,activating apoptosis and senescence-related procedures,thereby limiting this process,leading to cell cycle arrest and apoptosis.2.The level of MDM2 was decreased in the cell reprogramming process.The expression of p53 was significantly increased by MDM2 antagonist,which could increase the induction efficiency by the signal pathway of MDM2-p53 signal pathway.In the experiment,the expression of Aurka was inhibited by VX-680,but the level of Aurka was up-regulated during the cell reprogramming process.We found that the level of p53 was also up-regulated.Therefore,in the process of cell reprogramming,Aurka Not in the p53 signaling pathway plays a major role,but because of a number of factors together. |
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