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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 observe the effects of HSP70 small interfering RNA (siRNA) on the proliferation and apoptosis of cervical cancer HeLa cells in vitro and to explore the function of HSP70 in the proliferation and apoptosis of HeLa cells. Methods The siRNA sequence of HSP70 gene was designed and cloned into empty vector pTZU6 + 1. The recombinant plasmid was transformed into DH5α and the plasmid was extracted for enzyme digestion and sequencing analysis. HeLa cells were transfected with Lipofectamine 2000, while empty vector was transfected into negative control and blank control without any reagents. 48h after transfection, the expression of HSP70 mRNA and protein in HeLa cells was detected by semi-quantitative RT-PCR and Western blot; the growth inhibition rate of HeLa cells was detected by MTT assay; the morphological changes of HeLa cells were observed by AO / EB staining; HeLa cell apoptosis rate and cell cycle distribution. Results Compared with the control group, the mRNA and protein expression of HSP70 in transfected pHSP70-siRNA group were significantly decreased (P <0.01), indicating that transfection of Phsp70-siRNA plasmid can effectively reduce the expression of HSP70; the cell growth inhibition rate was significantly increased (P <0.05), and the cell inhibition rate reached the maximum at 48h. The apoptotic morphology of HeLa cells was observed under a fluorescence microscope. The results of flow cytometry showed that the apoptosis rate of HepG2 cells was significantly higher than that of the control group, G1 phase cells decreased, G2 / M and S phase cells increased. Conclusion HSP70 siRNA can effectively inhibit the proliferation of HeLa cells and promote the apoptosis of HeLa cells. HSP70 may be a new target of cervical cancer gene therapy.