【摘要】 目的筛选可抑制HeLa细胞p42MAPK表达的siRNA。方法采用体外转录合成的方法,合成针对p42MAPK的siRNA3条和阴性对照siRNA1条,并进行Cy-3荧光标记,用脂质体转染HeLa细胞,以判断转染效果。应用噻唑蓝(MTT)法和流式细胞仪检测siRNA的作用效果,筛选具有功能的siRNA序列;应用Westernblot方法鉴定siRNA抑制p42MAPK表达的效果。结果从3条p42MAPKsiRNA中筛选出2条有效的序列siRNA-2和siRNA-3,与空白对照组、脂质体对照组和随机siRNA-4相比,两者均可诱导HeLa细胞p42MAPK表达下调,Westernblot结果分析显示,与随机siRNA-4相比分别下调了1/2和2/3,并抑制HeLa细胞的增殖(P<0·05),同时改变了细胞周期各时相的细胞MAPK分布。结论p42MAPKsiRNA-2和siRNA-3在体外实验中可沉默目的基因以及抑制HeLa细胞增殖。更多还原

【Abstract】 Objective To screen for siRNAs that inhibit the expression of p42 MAPK in HeLa cell line. Methods Three p42 MAPK siRNAs and one random siRNA were synthesized using Silencer TM siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin TM 2000. The expression of p42 MAPK was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry. Results Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42 MAPK expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control. Conclusion Inhibition of p42 MAPK expression with siRNA technique can inhibit the proliferation of HeLa cells.更多还原