南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (08): 1118-1120.

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RNA干扰热休克蛋白90α表达对NIH-3T3细胞应激反应的影响

陈雪梅； 邹飞；   

  1. 南方医科大学劳动与职业卫生学系； 南方医科大学劳动与职业卫生学系 广东广州510515； 广东广州510515；
  • 出版日期:2006-08-20 发布日期:2006-08-20
  • 基金资助:
    国家自然科学基金(30371575,30500580);广东省自然科学基金(05300465)~~

Stress response changes of NIH-3T3 cells with HSP90α expression inhibition by RNA interference

CHEN Xue-mei, ZOU Fei Department of Occupational Hygiene, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学劳动与职业卫生学系； 南方医科大学劳动与职业卫生学系 广东广州510515； 广东广州510515；
  • Online:2006-08-20 Published:2006-08-20

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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抑制表达对细胞应激反应的影响。方法将人Hsp90αRNA干扰基因片段连接入pSilencer2.1-U6neo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,用免疫荧光、免疫印迹鉴定阳性克隆。用高温作用(44℃,40min)模拟氧化应激环境,以NIH-3T3细胞为对照,流式细胞仪测定DNA受损细胞数,分析在细胞应激状态下对细胞膜和DNA的损害及HSP90低表达对细胞应激反应的影响。结果转染pSilencerHSP90的NIH-3T3细胞Hsp90免疫荧光染色减弱,主要分布于胞浆;与对照相比,44℃,40min时DNA的损害加重(9.2%vs18.5%,P<0.01)。结论建立稳定低表达HSP90αNIH-3T3细胞株;热休克蛋白90表达与其应激保护作用密切相关。 

Abstract: Objective To establish a heat shock protein 90α(HSP90α) expression-inhibited cell line and study the effect of lowered HSP90α level on cell stress response. Methods The recombinant plasimid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90α was subcloned, purified and identified by DNA sequence analysis before introduced into mouse fibroblast cell line NIH-3T3 by electroporation. After G418 selection, the positive clones were identified by immunofluorescence and Western blotting. NIH-3T3 cells were subjected to hyperthermia at 44 ℃ for 40 min to simulate oxidative stress, and flow cytometry was performed to analyze the effect of low-level HSP90 on DNA damage under stress condition. Results Immunofluorescence and Westen blotting showed lowered HSP90 levels in the transfected cells. Compared with the control cells, cells subjected to hyperthermia displayed intensified DNA damage. Conclusion Low-level HSP90α causes the cells to be more vulnerable to oxidative stress condition, and HSP90 content can be associated with cell protection against such condition. 

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