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。
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 SilencerTM siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfeeted into HeLa cells by LipofectaminTM 2000. The expression of p42^MAPK was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTr 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.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2006年第5期292-295,共4页
Chinese Journal of Pathology
基金
陕西省自然科学基金项目(2003C215)
