The Impact Of Regulating DNMT1on Fetal Bovine Fibroblast Growth, Cycle, Apoptosis

Somatic cell nuclear transfer (SCNT) as a well developed and increasingly sophisticated technology, plays an important role in many fields. However, low efficiency has become the biggest obstacle in its development. Abnormal epigenetic modification is considered one of the main causes of low efficiency of SCNT. Dnmtl plays an important role in maintaining genomic methylation, and it is very important to gene expression of cloned embryos.In this study, we used different concentrations of5-aza-2’-deoxycytidine treated fetal bovine fibroblasts and designed three different fragments Dnmtl specific siRNA transfected fetal bovine fibroblasts, to evaluate different treatment on fetal bovine fibroblast proliferation ability, cell viability, cell cycle, apoptosis, then detect Dnmtl expression levels in fetal bovine fibroblasts. In order to detect the effects of different treatment on fetal bovine fibroblasts, providing reference to Dnmtl research and SCNT donor cells selection. The main findings are as follows:1.The effect of different concentrations of5-aza-CdR on proliferation activity, cycle, apoptosis and Dnmtl expression levels of fetal bovine fibroblast.Selected different concentrations of5-aza-2’-deoxycytidine (0,0.01,0.1,0.5,1μmol/L) treated fetal bovine fibroblasts. The results showed that, after96h continuous determination, compared with the control group, the proliferation rate and cell viability of the experimental group was decreased through time;24-96h, higher concentrations (0.5,1μmol/L) of5-aza-CdR has a significantly inhibition on fetal bovine fibroblast proliferation (p﹤0.05), and at48-72h cell viability decreased significantly (p<0.05).24-72h, treated fetal bovine fibroblasts with0.5and1μmol/L5-aza-CdR, cell cycle can significant inhibitied into the G0/G1phase (p<0.05); In addition to treated fetal bovine fibroblasts with0.01μmol/L5-aza-CdR has not significant influence to apoptosis, compared with control group, the remaining groups has a statistical significant influence to apoptosis (p<0.05). After48h0.5and1μmol/L of5-aza-CdR treatment, Dnmtl expression levels of fetal bovine fibroblasts were decreased14%and24%. In summary, although treated fetal bovine fibroblasts with the higher concentrations of5-aza-CdR can decrease Dnmtl expression levels, but it has disadvantage impact to fetal bovine fibroblasts growth, only less than0.1μmol/L5-aza-CdR is suitable for treated fetal bovine fibroblasts. 2.The effect of different Dnmtl specific siRNA fragments transfected fetal bovine fibroblasts on proliferation activity, cycle, apoptosis and Dnmtl expression levels of fetal bovine fibroblast.Designed three different fragments specific siRNA transfection Dnmtl fetal bovine fibroblasts. The results showed that:24h,48h,72h, compared with the control group, Dnmtl-siRNA3has a highly significant inhibition to Dnmtl expression (p<0.01), and at the time of48h, Dnmtl expression level decreased to nearly80%; compared with the control group, Dnmtl-siRNA3has a statistical significant inhibition to fetal bovine fibroblast proliferation (p<0.05), and cell viability decreased significantly (p<0.05). Dnmtl-siRNA3can obstruct cell cycle in G0/G1phase (p<0.05). Dnmtl-siRNA3significantly induced apoptosis (p﹤0.05). Thus, knowndown Dnmtl can induce abnormal cell proliferation, cell viability decreased, cell cycle arrest and apoptosis.