CREG基因沉默诱导小鼠ESCs来源的EBs自发性凋亡  

CREG gene silencing induces spontaneous apoptosis of ESCs-derived EBs

作　　者：张娜[1] 韩雅玲[1] 田孝祥[1] 李杰[1] 彭程飞[1] 闫承慧[1] 

机构地区：[1]沈阳军区总医院,全军心血管病研究所,辽宁沈阳110016

出　　处：《中国病理生理杂志》2011年第8期1574-1579,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30971218No.81004653No.81008097)

摘　　要：目的:探讨E1A激活基因阻遏子(CREG)基因沉默诱导鼠源性胚胎干细胞(ESCs)来源的胚胎小体(EBs)自发性凋亡的作用。方法:应用pDS-shRNA-CREG逆转录病毒真核表达载体和绿色荧光蛋白(GFP)的对照载体,感染鼠源性胚胎干细胞R1,筛选获得稳定的细胞克隆(R1-shCREG和R1-GFP);用小鼠胚胎成纤维细胞株STO作为饲养细胞,进行R1、R1-shCREG和R1-GFP细胞培养。倒置显微镜观察3种ESCs的生长情况。碱性磷酸酶(AKP)染色和小鼠心肌接种畸胎瘤形成实验检测转染后ESCs分化情况。当ESCs生长至亚融合状态时进行传代,去除白血病抑制因子(LIF)并悬浮培养制备EBs。用RT-PCR技术和Western blotting方法分别检测3组EBs培养7 d时CREG和cleaved caspase-3蛋白和mRNA的表达,Annexin V/PI流式细胞术检测EBs细胞凋亡情况。结果:经pDS-shRNA-CREG逆转录病毒真核表达载体及GFP的质粒载体转染获得稳定转染的细胞克隆。AKP染色证实R1、R1-GFP和R1-shCREG 3组ESCs均保持未分化状态。同时,体内和体外分化研究证实,R1和R1-GFP组细胞具有三胚层分化能力,小鼠心肌内注射的ESCs形成畸胎瘤组织;而R1-shCREG则不能够形成畸胎瘤类似物,细胞分化能力降低,且细胞死亡现象增加。培养7 d的R1-shCREG/EB组CREG蛋白表达量较R1/EB组和R1-GFP/EB组均降低;CREG的mRNA表达下降;而cleaved caspase-3 mRNA和蛋白表达均显著升高;同时,R1-shCREG/EB细胞凋亡率明显升高。结论:下调CREG表达可抑制ESCs分化,促进ESCs来源的EBs细胞凋亡的发生。AIM： To investigate the effect of cellular repressor of E1A - stimulated genes （CREG） on the spontaneous apoptosis of murine embryonic stem cells （ESCs） - derived embryoid bodies （EBs）. METHODS ： The murine ESCs R1 were infected with pDS - shRNA - CREG and pDS - shRNA - GFP retrovirus, respectively. R1, R1 - GFP and R1 - shCREG were cultured on STO feeder ceils in DMEM supplied with leukemia inhibitory factor（LIF）. Alkaline phosphatase（AKP） staining and teratoma formation assay inoculated into mouse myocardium were used to detect stemness of transfected ESCs. R1/EB, R1 - GFP/EB and R1 - shCREG/EB were produced by liquid suspension method. The expression of CREG and cleaved caspase - 3 were analyzed by Western blotting and quantitative RT - PCR on day 7. The apoptot- ic rates of the 3 kinds of EBs were analyzed by flow cytometry （FCM） analysis with Annexin V/PI dual staining. RESULTS： The stably -transfected ES cells （R1 -shCREG and R1 -GFP） were obtained by screening the G418 -resistant clones. R1 - GFP and R1 - shCREG had the stem cell properties similar to those of R1 detected by AKP staining. However, it was found that R1 - shCREG didn＇t show the same almighty differentiation function as of R1 and R1 - GFP by tumor experiments in mouse myocardium that it couldn＇t form teratomas analogue and had the lower ability of cell differentiation. Observation under phase - contrast microscope showed that the cell differentiation was inhibited while the number of cell death was increased in R1 - shCREG/EB group. Compared with R1/EB and R1 - GFP/EB, Western blotting analysis demonstrated that the protein expression of CREG was decreased to （78. 0 ±1.3 ） % and （ 84.0 ±2. 4） % on day 7, respectively. The mRNA expression of CREG was also decreased, but the expression of cleaved caspase - 3 at the mRNA and protein levels was increased obviously. Annexin V/PI FCM assay indicated that the apoptotic rate of R1 - shCREG/EB was significantly higher those that in other 2 groups on day

关 键 词：EIA激活基因细胞阻遏子 胚胎干细胞 细胞凋亡 胚胎小体 

分 类 号：R363[医药卫生—病理学]