【摘要】 目的 探讨小分子干扰RNA下调缺氧诱导因子-2α基因表达对人肝癌细胞株HepG2体外凋亡的影响,并研究其可能的分子机制。方法 采用不同浓度的氯化钴诱导细胞模拟缺氧,设计合成的特异性缺氧诱导因子-2αsiRNA转染缺氧环境下HepG2细胞48 h后,采用逆转录聚合酶链式反应、免疫蛋白印记方法 检测细胞中缺氧诱导因子-2αmRNA和蛋白表达,流式细胞术观察细胞凋亡率,半胱氨酰天冬氨酸特异性蛋白酶-9试剂盒检测细胞中半胱氨酰天冬氨酸特异性蛋白酶-9活性;免疫蛋白印记法检测凋亡相关因子B淋巴细胞/白血病-2、白血病-2相关x蛋白的表达。结果 低氧环境下,缺氧诱导因子-2α表达上调;特异性缺氧诱导因子-2αsiRNA转染HepG2细胞48 h后,转染组缺氧诱导因子-2αmRNA和蛋白表达下调(P<0.05);流式细胞术显示细胞凋亡率升高(P<0.05),活性检测显示半胱氨酰天冬氨酸特异性蛋白酶-9活性升高,免疫蛋白印记法检测显示白血病-2表达下调,x蛋白表达上调(P<0.05)。结论 特异性缺氧诱导因子-2αsiRNA可针对性的下调HepG2细胞中缺氧诱导因子-2α的表达,促进HepG2细胞体外凋亡,这一过程可能与下调白血病-2表达、上调x蛋白的表达及半胱氨酰天冬氨酸特异性蛋白酶-9活性有关。更多还原

【Abstract】 Objective To investigate the effect of siRNA down regulating of HIF-2 a gene expression on HepG2 apoptosis in vitro, and study its possible molecular mechanism. Methods Different concentrations of cobalt chloride were used to induce cell hypoxia. Using designed specific HIF-2 a siRNA to transfect HepG2 cells under hypoxia environment for 48 h, then Real-time PCR, Western blot methods were used to detect the HIF-2 a mRNA and protein expression, flow cytometry to observe the apoptosis rate, CaspaseGlo○R-9 kit to detect the activity of cysteinyl aspartate specific protease(Caspase)-9 in cells; and Western blot was used to detect the FAS B cell/Bcl-2, Bax expression. Results HIF-2 a expression raised in low oxygen environment; specific siRNA transfection of HepG2 cells after 48 h, HIF-2 a mRNA and protein expression of transfection group were down-regulated(P<0. 05); flow cytometry showed increase of apoptosis rate(P<0. 05), the activity test showed increased activity of Caspase-9, western blot showed reduced expression of Bcl-2 and increased expression of Bax protein(P<0. 05). Conclusion Specific HIF-2 a siRNA can down regulate HIF-2 a expression in HepG2 cells to promote the apoptosis of HepG2 cells in vitro. This process may be related to down regulation of Bcl-2 expression, up regulation of Bax expression and Caspase-9 activity.更多还原