摘要
目的:视网膜母细胞瘤样蛋白2(Rbl-2)在调控DNA甲基化转移酶(DNMT)中发挥重要作用,而DNMT影响干细胞的分化。本文旨在探讨调控Rbl-2基因的表达对心脏干细胞凋亡的影响。方法:分离培养人心脏干细胞,转染Rbl-2 siRNA。采用real time RT-PCR检测Rbl-2和DNMT-3B mRNA表达水平,Westernblotting检测DNMT-3B蛋白表达和caspase-3活化片段,并以Annexin V-FITC/PI双染色-流式细胞术检测细胞凋亡率。结果:在人心脏干细胞中,转染siRNA后Rbl-2 mRNA表达水平与阴性对照组相比显著降低(P<0.01),同时DNMT-3B mRNA和蛋白表达水平与阴性对照组相比均显著升高(P<0.01),差异显著。与阴性对照组相比,转染Rbl-2 siRNA的人心脏干细胞caspase-3激活,表现为caspase-3活性片段与caspase-3前体的比值显著增高(P<0.01),annexin V阳性细胞凋亡率上升(P<0.05)。结论:Rbl-2基因在心脏干细胞的生存中具有正性调控作用,抑制其表达能促进心脏干细胞的凋亡,其调控机制与表观遗传学的修饰密切相关。
AIM：Retinoblastoma-like protein 2（Rbl-2） plays an important role in the cell proliferation and DNA methyltransferase（DNMT） may involve in the regulation of differentiation in embryonic stem cells.This study is to investigate the effect of knocking down Rbl-2 by specific siRNA on apoptosis in human cardiac stem cells.METHODS： The siRNA of Rbl-2（siRbl-2） was transfected into human cardiac stem cells.The mRNA expression of Rbl-2 and DNMT-3B was detected by real-time RT-PCR 48 h after transfection.The DNMT-3B protein expression and the activation of caspase-3 were determined by Western-blotting.The cell apoptosis was analyzed by flow cytometry with annexin V-FITC/PI staining.RESULTS： Knocking down of Rbl-2 gene increased the expression of DNMT-3B in human cardiac stem cells,and induced cell apoptosis.Compared with negative control group,caspase-3 was activated and cleaved caspase-3 was increased in the stem cells transfected with siRbl-2.The cleaved caspase-3 accounted for more proportions of total caspase-3 in transfected cells than that in non-transfected cells（P0.01）.The apoptotic rate was also increased significantly in transfected group（P0.01）.CONCLUSION： Rbl-2 plays an important role in the regulation of survival and apoptosis in human cardiac stem cells.This regulatory mechanism may involve in epigenetic modification,which is mediated by DNMT.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第12期2306-2310,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30772142
No.81070103)
