HSP90高表达细胞株的建立及细胞增殖性状的改变

目的:建立稳定高表达热休克蛋白90(HSP90)细胞株,研究其对细胞增殖的影响.方法:含人HSP90 13全长基因的重组质粒pSmycHSP经亚克隆、纯化、酶切鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内.经G418筛选、克隆分离培养,用免疫细胞化学、免疫印迹鉴定阳性克隆.以转染空质粒的NIH-3T3细胞为对照,用MTT法、流式细胞术测定,分析HSP90高表达对细胞增殖和细胞周期的影响.结果:转染pSmycHSP的NIH-3T3细胞HSP90染色增强,生长速度减慢,S期DNA含量降低.结论:己建立稳定高表达热休克蛋白90(HSP90)NIH-3T3细胞株;转染pSmycHSP的NIH-3T3细胞能够有效地表达HSP90,影响细胞周期,使细胞增殖迟滞.

Establishment of HSP90 overexpressing cell line and effects on cell proliferation

Aim: To establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response.Me thods: The recombined plasmid pSmycHSP,which contained the full length DNA coding for human HSP90 B,was introduced into mouse fibroblast cell line NIH 3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion.After screened by G418,the positive clones were selected and identified by immunocytochemistry and Western blotting.Contrasted with NIH 3T3 cells transfected with empty plasmid,the effect of high level HSP90 on cell proliferation and cell cycle was analyzed by MTT method and flow cytometry.Results: The rising level of HSP90 was shown by immunocytochemistry and Western blotting.Compared with control,the growth of HSP90 highly expressing cell line slowed down and the DNA content of S phase was lower.Conclusion: The NIH 3T3 derived cell line,which stably expressed high level of HSP90 was established.The effect of high level HSP90 on cell prolife ration was to retard cell growth by affecting cell cycle.