磷酸酶与张力蛋白同源物基因过度表达抑制乳鼠心脏成纤维细胞增殖  

Overexpression of PTEN inhibits proliferation of cardiac fibroblasts in neonatal rats

作　　者：聂凌[1] 祝善俊[1] 于林君[1] 宋熔[1] 王江[1] 田颖[1] 

机构地区：[1]第三军医大学新桥医院心血管内科,重庆400037

出　　处：《中华老年心脑血管病杂志》2007年第4期262-265,共4页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家自然科学基金资助项目(30370584)

摘　　要：目的将携带有张力蛋白同源物基因(PTEN)的重组腺病毒载体转染乳鼠心脏成纤维细胞,观察心脏成纤维细胞过度表达野生型PTEN对其增殖的影响。方法分离培养心脏成纤维细胞,以携带有绿色荧光蛋白(GFP)的重组腺病毒Ad-PTEN转染上述细胞,荧光倒置显微镜下观察GFP表达情况,确定最佳感染倍数(MOI)。原代心脏成纤维细胞接种至96孔板,转染Ad-PTEN[Ad-PTEN转染组(MOI根据生长曲线确定)],并设正常对照组(加入等体积培养基),以MTT的方法检测其吸光度(A),以反映细胞增殖情况,将转染效率达到90%以上成纤维细胞及正常对数生长期成纤维细胞消化,制成单细胞悬液,流式细胞仪分别检测细胞周期,以吖啶橙/溴乙锭(AO/EB)法分别检测Ad-PTEN转染组和正常对照组细胞凋亡情况;结果MTT测定的结果:48 h后Ad-PTEN转染组的A显著低于该时间点正常对照组(P<0.05)。流式细胞仪检测结果发现Ad-PTEN转染的成纤维细胞出现G1期阻滞;AO/EB法检测发现Ad-PTEN转染的成纤维细胞其细胞凋亡率与正常对照组细胞无显著差异;结论PTEN基因过度表达将引起乳鼠心脏成纤维细胞增殖受抑制,该现象与细胞周期G1期阻滞有关。Objective To observe the effect of overexpression of PTEN by transfection of recombinant adnovirus containing wild-type PTEN on proliferation of cardiac fibroblasts in neonatal rats. Methods Cardiac fibroblasts were isolated, cultured and transfected by Ad-PTEN containing green fluorescent protein（GFP）. The most suitable multiple of infecfion（MOI） was determined by observing the expression of GFP. Cardiac fibroblasts were inoculated into 96-well plate and tranfected by Ad-PTEN（MOI was detennined by growth curve）. Absorbency of each well was measured by MTT. Cell cycle of transfected cardiac fibroblasts and the control cells were measured by flow cytometry. Apoptosis rates of these two groups were measured by method of AO/EB. Results The absorbency of tranfected fibroblasts was significantly lower than that of the control cells（ P 〈 0.05）after 48 hours. G1 arrest was observed in the transfected cells by flow cytometry. There was no significant difference in apoptosis rate between these two groups. Conclusion Overexpression of PTEN gene will inhibit the proliferation of cardiac fibroblasts because of G1 arrest in neonatal rats.

关 键 词：心内膜心肌纤维化症 磷酸单脂水解酶类 细胞凋亡 基因表达 转染 

分 类 号：R541[医药卫生—心血管疾病]