低氧诱导因子-1a受体的小干扰RNA对人脊髓星形胶质瘤细胞凋亡的影响
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摘要
目的 研究低氧诱导因子-1α(HIF-1α)受体的小干扰RNA对人星形胶质瘤细胞(U251)凋亡的影响，小干扰RNA对HIF-1α的调控作用；探讨肿瘤细胞凋亡的分子机制及调控。
方法 构建靶向低氧诱导因子-1α(HIF-1α)受体的小干扰RNA(si RNA)真核表达载体pSUPER EGFP1。培养成熟的U251细胞株随机分成：①、载体(载体中转染HIF-1α基因)缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)：②、载体常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)；③、空载体(载体中未转染HIF-1α基因)缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)；④、空载体常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)；⑤、对照缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)；⑥、对照常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)。每一个样品采用real time PCR测定HIF-1αmRNA表达水平；Western Blot和免疫组织化学测定HIF-1α蛋白表达水平；光镜和电镜分别观察细胞、细胞线粒体和足突改变；荧光显微镜观察细胞荧光；
Objective To investigate the small interfering RNA of HIF-1α receptor effects the human U251 cell apoptosis, and regulation of HIF-1α expression by small interfering RNA, and use of PDTC(PDTC group), and to explore the molecular mechanism of tumor cell apoptosis and its regulation.
Methods The small interfering RNA(siRNA) eukaryotic expression vector targeting HIF-1α gene, named pSUPER EGFP1, was constructed and identified with sequencing. After the siRNA expression vector was transfected into the human U251 cells, The cultured human U251 cells were randomly assigned to six group:①vector hypoxia group(HIF-1α gene transfect into the vector )(the human U251 cells was exposed to 1%O_2, without FBS and glucose for 12h,24h and 48h respectively);②vector nomal O_2 group(HIF-1α gene transfect into the vector )(the human U251 cells was exposed to nomal O_2, without FBS and glucose for 12h,24h and 48h respectively);③empty vector hypoxia group(HIF-1α gene nontransfected
方法 构建靶向低氧诱导因子-1α(HIF-1α)受体的小干扰RNA(si RNA)真核表达载体pSUPER EGFP1。培养成熟的U251细胞株随机分成：①、载体(载体中转染HIF-1α基因)缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)：②、载体常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)；③、空载体(载体中未转染HIF-1α基因)缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)；④、空载体常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)；⑤、对照缺氧组(U251在1％氧，无糖和血清的培养基中分别培养12h、24h和48h)；⑥、对照常氧组(U251在常氧，无糖和血清的培养基中分别培养12h、24h和48h)。每一个样品采用real time PCR测定HIF-1αmRNA表达水平；Western Blot和免疫组织化学测定HIF-1α蛋白表达水平；光镜和电镜分别观察细胞、细胞线粒体和足突改变；荧光显微镜观察细胞荧光；
Objective To investigate the small interfering RNA of HIF-1α receptor effects the human U251 cell apoptosis, and regulation of HIF-1α expression by small interfering RNA, and use of PDTC(PDTC group), and to explore the molecular mechanism of tumor cell apoptosis and its regulation.
Methods The small interfering RNA(siRNA) eukaryotic expression vector targeting HIF-1α gene, named pSUPER EGFP1, was constructed and identified with sequencing. After the siRNA expression vector was transfected into the human U251 cells, The cultured human U251 cells were randomly assigned to six group:①vector hypoxia group(HIF-1α gene transfect into the vector )(the human U251 cells was exposed to 1%O_2, without FBS and glucose for 12h,24h and 48h respectively);②vector nomal O_2 group(HIF-1α gene transfect into the vector )(the human U251 cells was exposed to nomal O_2, without FBS and glucose for 12h,24h and 48h respectively);③empty vector hypoxia group(HIF-1α gene nontransfected
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