| Embryonic stem cells(ESCs)are derived from the inner cell mass(ICM)of the developing blastocyst.ESCs can proliferate infinitely and stay in an undifferentiated state,which is called self-renewal.ESCs have potential to differentiate into any type of cells in the three germ layers.Embryonic stem cells were injected into embryo can form chimera,and develop into various adult tissues.ESCs can maintain undifferentiated proliferation state and have the potential to differentiate into specific cell types while cultured and passaged in vitro.ESCs proliferate more rapidly than the regular differentiated cells,upregulating the gene mutation probability,hence maintaince of the genomic integraty becomes more important.ESCs stayed near the apoptotic threshold to get ready for the elimination of the mutated cells.Therefore,self-renewal of ESCs is an equilibrium mechanism of proliferation and apoptosis,is crucial for understanding the developmental process.p57 is a member of the cyclin-dependent kinases inhibitory proteins(CKIs)which can prevent the formation of complexes between cyclin-dependent kinases(CDKs)and cyclins in the G1/S phase,and then inhibit the phosphorylation of Rb as a key negative regulatory role of cell cycle.p57 aberrant expression can lead to lesions in the early embryonic development of humans and mice.Research on the mechanisms of p57 regulating self-renewal in embryonic stem cells is rare.p57 modulates cell cycle widely,and influences the apoptosis by inhibition of cell proliferation,its effects on the apoptotic pathway are closely related with the carcinogenesis.Mainly through the perturbance of p57 expression,we detected the influences on the cell cycle and apoptosis,and then combined the results to study their effects on the self-renewal of the ESCs.Perform tentative test of p57 effects on the pluripotent gene and the related DNA methylation.In this study,p57 shRNA interference vector was constructed,and transfected into mouse ESCs leading to p57 down-regulation.Through examination and analysis of proliferation,apoptosis and pluripotency,we have explored how p57,a cell cycle negative regulator protein,affected the self-renewal of mouse ESCs and the underlying regulatory mechanisms.1.ESCs proliferation was promoted by p57 interferenceBy measuring the area of ESCs clones,it was found that down-regualtion of p57 promoted the growth of ESCs.This was further proved by the growth curve of the cells.The ESCs that interfered of the p57 were able to synchronize with the control group at the logarithmic phase and had a similar proliferation rate.However,it can delay the entry into the plateau and decline period of cell generation cycle.Edu detection showed that interference of p57 could increase the positive rate of Edu staining of ESCs and increase proliferation.Cyclin A,cyclin E,and cyclin D showed no significant changes,and cyclin kinase Cdk2 retained the high expression level as the control group,but it did not increase significantly.Western Blot showed significant upregulation of PCNA.With the detection of new pluripotent genes in cells,Oct4 and Zfp42 were significantly up-regulated at the mRNA level.When coming to protein level,Klf4 was significantly up-regulated in the interfering group ESCs,Ezh2 and its modificated H3K27me3 were significantly upregulated,indicating that interference p57 can enhance the proliferation potential and pluripotency of ESCs,which is independent of the traditional cell cycle regulatory system.2.ESCs interfered of p57 inhibited apoptosis occuranceTUNEL staining revealed that there was a decrease in the rate of apoptotic cells in the interfering group.Annexin V/PI detection revealed that the rate of late-stage apoptotic cells(double stained)in clones was relatively decreased in interfering group.By RT-qPCR detection,p21 in the interfering group was significantly down-regulated,while the ratio Bcl2/Bax was significantly up-regulated.Western Blot showed that the expression of phosphorylated p53 was up-regulated in the interfering group and p53 expression was down-regulated correspondingly,indicating that p53 activity was inhibited.Bax(pro-apoptotic protein)was significantly down-regulated,while Caspase 3 and its cleaved Caspase 3 were down-regulated.In conclusion,it was shown that after interference of p57,the pluripotency of ESCs was enhanced,which in turn reduced the probability of ESCs apoptosis. |
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