HSP70 siRNA对宫颈癌HeLa细胞HSP70表达及细胞增殖与凋亡的影响  

Effect of HSP70 siRNA on expression of heat shock protein 70 in human cervical cancer Hela cells and their proliferation and apoptosis

作　　者：韩建红[1] 赖国旗[1,2] 陈妮[1] 易发平[3] 黄爱龙[2] 

机构地区：[1]重庆医科大学实验动物中心,重庆400016 [2]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016 [3]重庆医科大学分子生物学教研室,重庆400016

出　　处：《第三军医大学学报》2010年第4期342-345,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30800945);重庆市自然科学基金(CSTC2008BB5235)~~

摘　　要：目的通过体外实验观察HSP70的小分子干扰RNA(small interfering RNA,siRNA)对宫颈癌HeLa细胞株增殖及凋亡的影响,探讨HSP70在HeLa细胞增殖和凋亡中的功能。方法针对HSP70基因设计siRNA序列,克隆到空载体pTZU6+1中,转化DH5α菌株,提取质粒,进行酶切鉴定和测序分析。在脂质体2000的介导下转染HeLa细胞,同时转染空载体为阴性对照及不加任何试剂的空白对照。转染48h后,应用半定量RT-PCR,Westernblot检测HeLa细胞HSP70mRNA及蛋白的表达情况;MTT法检测HeLa细胞的生长抑制率;AO/EB染色法观察HeLa细胞形态学变化;流式细胞术检测HeLa细胞的凋亡率和细胞周期分布情况。结果与对照组相比,转染pHSP70-siRNA组HSP70的mRNA及蛋白表达均明显下降(P<0.01),表明Phsp70-siRNA质粒转染能有效降低HSP70的表达;细胞生长抑制率明显增高(P<0.05),且在48h时细胞抑制率达到最大;在荧光显微镜下观察到HeLa细胞出现凋亡形态;流式细胞术结果显示pHSP70-siRNA组细胞凋亡率显著高于对照组,且G0/G1期细胞减少,G2/M和S期细胞增多。结论HSP70siRNA可以有效抑制宫颈癌HeLa细胞增殖,促进HeLa细胞凋亡;HSP70可能成为宫颈癌基因治疗的一个新靶点。Objective To study the function of heat shock protein 70 （HSPT0） on proliferation and apoptosis of HeLa cells by observing the effect of its small interfering RNA （siRNA） on them in vitro. Methods siRNA targeting HSP70 gene was designed and cloned into the vector pTZU6 ＋ 1 （pHSP70-siRNA） and transformed into DHSa followed by extraction of plasmid DNA. pHSP70-siRNA was identified by restriction enzyme digestion and sequencing for analysis and transfected into HeLa cells with LipofectamineTM 2000. The transfected pTZU6 ＋ 1 was used as a negative control and the non-transfected pTZU6 ＋ 1 served as a blank control. After 48 hours of transfection, the expression levels of HSP70 mRNA and protein were measured by semi-quantity RT-PCR and Western blotting, respectively. The growth inhibition ratio of HeLa cells was determined by MTT. AO/EB staining was used for observation of morphologic changes in HeLa cells. The apoptosis rate and cell cycle were detected by flow cytometry. Results The expression levels of HSP70 mRNA and protein were significantly lower while the cell growth inhibition rate was significantly higher in HeLa cells than in negative controls （ P 〈 0.01, 0.05 ）. Apoptotic morphology was observed in HeLa cells under a fluorescence microscope. Flow cytometry showed that the apoptosis rate was significantly higher in pHSP70-siRNA than in controls with the cells arrested mainly in G2/M phase. Conclusion HSP70-siRNA can effectively inhibit the proliferation and promote the apoptosis of HeLa cells, and may thus become a new target of gene therapy for cervical cancer.

关 键 词：HSP70热休克蛋白质类 RNA小分子干扰 HELA细胞 细胞凋亡 

分 类 号：R394.2[医药卫生—医学遗传学] R73-36[医药卫生—基础医学]