摘要
目的用RNA干扰方法建立稳定的热休克蛋白90α(HSP90α)抑制表达细胞株,研究热休克蛋白90α抑制表达对人肝癌HepG2细胞生物学行为的影响。方法将人HSP90αRNA干扰基因片段连接入pSilencer 2.1-U6 neo载体,重组质粒pSilencerHSP90经亚克隆、纯化、测序鉴定后,用电穿孔法转染到人肝癌细胞系HepG2细胞内。经G418筛选后,阳性克隆被进一步分离培养,形成稳定的HSP90α抑制表达细胞株。将此细胞株作为siRNA干扰组,将未转染的HepG2细胞作为对照组。用荧光定量PCR,免疫印迹检测siRNA对HSP90α的抑制效果,采用CCK-8法测定其对细胞增殖的影响,流式细胞仪检测其对细胞周期的影响。结果测序表明HSP90α干扰序列完全正确;siRNA干扰组HSP90αmRNA水平降低,蛋白表达量也有明显的减少;与对照组相比,siRNA干扰组在48 h后可明显抑制细胞增殖,流式细胞术检测siRNA干扰组细胞有明显的G2/M期细胞周期阻滞。结论建立稳定低表达HSP90α的HepG2细胞株;下调HepG2细胞内HSP90α的基因表达可抑制细胞增殖,引起明显的细胞周期阻滞。HSP90α靶向siRNA干扰为肝癌的基因治疗提供了可能。
Objective To establish a cell line stably inhibiting the heat shock protein 90α（HSP90α）expression by siRNA interference and to explore the effect of HSP90α siRNA on proliferation and cell cycle of liver cancer cell line HepG2. Methods The recombinant plasmid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90α was subcloned,purified and identified by DNA sequence analysis.Then the recombinant plasmid pSilencerHSP90 was introduced into HepG2 cell by electroporation.After G418 selection,positive clones were isolated and cultured to form a cell line stably inhibiting HSP90α expression（siRNA interference group）.The non-transfected HepG2 cells were chosen as control group.The positive clones were identified by quantitative RT-PCR and Western blot.CCK-8 assay and flow cytometry（FCM） were applied to determine the cell growth and cell cycle,respectively. Results The sequence of specific siRNA was correct by sequence analysis.The levels of HSP90α mRNA and protein expression were reduced in siRNA interference group.The CCK-8 assay results showed that the cell growth in siRNA interference group was significantly inhibited after 48 h compared with control group.The FCM results showed that there was an obvious G2/M phase arrest in siRNA interference group. Conclusion The cell lines stably low expressing HSP90α is successfully established.The decreased expression of HSP90αgene can inhibit the cell proliferation,and cause the cell cycle arrest.Successful cloning of the recombinant plasmid may help to find a new gene therapy for hepatoma.
出处
《山西医科大学学报》
CAS
2011年第6期443-446,共4页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(305000580
30971193)
广东省自然科学基金资助项目(05300465)
