摘要
背景:已有研究发现,ROCK1和ROCK2参与了细胞的凋亡过程,由此假设下调ROCK1和ROCK2表达能抑制心肌细胞凋亡。目的:验证ROCK1-shRNA和ROCK2-shRNA转染能否有效沉默大鼠心肌细胞内ROCK1和ROCK2基因的表达,并筛选沉默效率最高的shRNA。方法:以ROCK1和ROCK2为靶基因,分别构建了3个特异性shRNA重组质粒,转染这些shRNA后观察其对SD大鼠心肌细胞中ROCK1和ROCK2的沉默作用,并筛选出沉默效率最佳的shRNA。结果与结论:WesternBlot结果显示,与对照组相比,3个重组质粒ROCK1-shRNA和ROCK2-shRNA的转染均能明显降低SD大鼠心肌细胞中ROCK1和ROCK2的表达(P<0.05或P<0.01),其中ROCK1-shRNA1和ROCK2-shRNA2的沉默效率最高(P<0.05),分别为(82.15±7.53)%和(89.42±5.81)%。这表明ROCK1-shRNA和ROCK2-shRNA转染能有效沉默大鼠心肌细胞内ROCK1和ROCK2基因的表达,并筛选出了沉默效率最高的shRNA。
BACKGROUND： Previous studies have proved that Rho-associated coiled-coil protein kinase （ROCK） 1 and ROCK2 involve in the process of apoptosis. On that basis, a hypothesis has been proposed that myocardial apoptosis can be inhibited by down-regulating the expression of ROCK1 and ROCK2. OBJECTIVE： To verify whether the transfection of ROCKl-short hairpin RNA （ShRNA） and ROCK2-shRNA can effectively silence the expressions of ROCK1 and ROCK2 in rat cardiomyocytes and to screen for the shRNA with the best silencing effeciency. METHODS： Three specific recombinant plasmids of ROCKI-shRNA and ROCK2-shRNA were constructed and identified respectively. Silencing effects of ROCK1 and ROCK2 in SD rat myocardial cells were assessed after shRNA transfection. And the shRNA with the best silencing efficiency was selected among these shRNAs. RESULTS AND CONCLUSIONS： Western blot results showed that, compared with the control groups, the transfection of all three recombinant plasmids of ROCKI-shRNAs and ROCK2-shRNAs inhibited the expressions of ROCK1 and ROCK2 significantly （P 〈 0.05 or P 〈 0.01 ）. Among these shRNAs, ROCKI-shRNA1 and ROCK2-shRNA2 showed the best silencing efficiency of （82.15±7.53）% and （89.42±5.81）%, respectively （P 〈 0.05）. These findings show that the expressions of ROCK1 and ROCK2 in rat cardiomyocytes can be effectively inhibited by the transfection of ROCKI-shRNA and ROCK2-shRNA. And the shRNA with the best silencing efficiency was selected among these shRNAs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2011年第50期9441-9444,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家重点基础研究发展"九七三"计划(2008CB517305)资助
课题名称:"ROCKs和TNNI3K基因在冠心病发病过程中作用的基础研究"
国家自然科学基金(81060013)资助
课题名称:"肌细胞缺氧损伤时唯BH3蛋白Bim的作用及转录调节"~~
