siRNA抑制缺氧诱导因子-1ɑ表达对人视网膜母细胞瘤细胞增殖与凋亡的影响  

Inhibition of hypoxia inducible factor 1α by siRNA-induced apoptosis in human retinoblastoma cells

作　　者：郎莉莉[1] 高玉[2] 葛茸茸 陈建梅[1] 景明[2] 

机构地区：[1]同济大学附属东方医院眼科,上海200120 [2]解放军第四一一医院眼科

出　　处：《海军医学杂志》2016年第1期22-26,共5页Journal of Navy Medicine

基　　金：上海市卫生和计划生育委员会科研资助课题(201540304);上海市虹口区卫生和计划生育委员会科研资助课题(虹卫1402-11)

摘　　要：目的观察si RNA抑制人视网膜母细胞瘤表达低氧诱导因子(HIF)对细胞增殖和凋亡的影响。方法以Y-79细胞系为研究对象,分别在正常氧和低氧环境下培养,MTT法检测细胞增殖,RT-PCR和Western blot检测HIF-1ɑm RNA和蛋白质水平的表达变化,si RNA抑制细胞表达HIF-1ɑ,膜联蛋白V-FITC和PI双染法流式细胞术检测细胞凋亡情况,荧光评估测定法检测半胱天冬酶活性。SPSS 16.0软件包作统计学分析,配对t检验。结果与常氧和低氧环境培养Y79细胞比较,在0、4、8、12 h后HIF-1ɑm RNA和蛋白质数倍高表达。si RNA干扰技术处理24、48、72 h后,常氧环境培养下的Y-79细胞表达HIF-1ɑ呈时间依赖性轻度下降趋势,低氧条件下呈显著下降。si RNA HIF-1ɑ可能是通过上调Bax/Bcl-2比率和活化caspase-9、caspase-3途径促进Y-79细胞凋亡。结论 si RNA HIF-1ɑ下调人视网膜母细胞瘤表达低氧诱导因子,可抑制细胞增殖和促进细胞凋亡。抑制HIF-1ɑ表达可能是治疗视网膜母细胞瘤的潜在新策略。Objective To investigate the effect of hypoxia inducible factor-la knocked down by small interfering RNA （siR- NA） on cell proliferation, apoptosis and apoptotic pathways of human Y-79 RB cells. Methods Cell proliferation, HIF-1α mRNA and protein levels were measured by MTT, RT-PCR and Western Blot on human Y-79 RB cells under both normoxie and hypoxic conditions, respectively, siRNA knockdown against HIF-1α was carried out to suppress the expression of HIF-1α. Cell apotosis was determined by double staining cells with the annexin V-FITC and PI. Caspase activity was assessed by the fiuorometric assay. Biostatistical analyses were conducted with SPSS 16.0 software package. Results A muhifold increase in HIF-1α mRNA and protein levels were observed af- ter cells were exposed to the hypoxic environment at 0 h,4 h,8 h and 12 h. Both mRNA and protein levels of HIF-1α were attenuated or abolished by transfeetion with siRNARIF-α under normoxic and hypoxic conditions. Futhermore, knockdown of HIF-1α could enhance hy- poxia-induced slight increase of Bax/Bcl-2 ratio and activate caspase-9 and caspase-3. Conclusion This study was able to show that a knockdown of HIF-la by siRNA, IF-1α resulted in a decrease in proliferation and induction of apoptosis in human Y-79 RB cells under both normoxic and hypoxic conditions. HIF-1 a expression may be a promising strategy for the treatment of human RB in the future.

关 键 词：低氧诱导因子-1ɑ 人视网膜母细胞瘤 细胞增殖 细胞凋亡 

分 类 号：R774[医药卫生—眼科]