细胞内钙在人胃癌细胞缺氧诱导因子1表达及转录激活中的作用论文.docVIP

　　细胞内钙在人胃癌细胞缺氧诱导因子1表达及转录激活中的作用论文 兰梅，时永全，韩者艺，宁晓喧，樊代明 【摘要】 目的观察细胞内钙变化对氧调节性亚单位HIF1α表达及其HIF1转录激活的影响。方法常氧时，采用通透性细胞内钙的螯合剂BAPTAAM或钙的离子载体Ionomycin降低或升高细胞内钙，用edicine, the Affiliated Hospital of Medical College of Shaoxing University, Shaoxing 312000, China; Institute of Digestive Disease State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University Abstract：Objective To study the possible roles of changed intracellular calcium level in SGC7901 cells on HIF1α expression and HIF1 transcriptional activity. MethodsUnder normoxic conditions, the protein expression of HIF1α in SGC7901 cells after treatment eant Ca2+ chelator), or ionomycin (an calcium ionophore) ycin on nuclear location of HIF1α, HIF1 luciferase activity as ined by indirect immunofluorescence assay, dual luciferase reporter system and enzymelinked immunosorbent assay (ELISA) respectively. ResultsDecreasing the intracellular calcium level by BAPTA induced HIF1α nuclear accumulation, stimulated HIF1dependent transcription and enhanced expression of VEGF in SGC7901 cells under normoxic conditions. Hoycin application, though it can mediated a ulation. ConclusionChelation of cellular calcium modulates HIF1α expression and HIF1 transcriptional activity in human gastric cancer cells. Changes in intracellular free calcium concentration appeared to have a critical role in hypoxic signal transduction in gastric cancer cells. Key ; HIF1; Protein expression; Transcriptional activity 0引言 近来发现1缺氧可使内皮细胞中的Ca2+明显增加，升高或降低细胞内Ca2+，可增加或降低缺氧诱导基因如血管内皮生长因子(vascular endothelial groycin及βactin的单抗为Sigma公司产品； HIF1α多抗为Santa公司产品； FuGENE6转染试剂购自Roche公司； 双荧光素酶报告基因检测试剂盒购自Promega公司； VEGF检测试剂盒购自上海森雄公司； pGL3SV40HRE质粒（在BamHⅠ位点插入2个拷贝的缺氧反应元件）为大阪大学医学院Mayuko Osada 教授惠赠。 1.2方法 1.2.1改变细胞内钙对HIF1α蛋白稳定的影响－ BAPTAAM、1μM ionomycin孵育0、2、4、8、12、24h，收获细胞。三去污裂解细胞制备总蛋白，Bradford法测定蛋白浓度；取100μg总蛋白上样，行SDSPAGE电泳，转膜，8%脱脂奶粉封闭2h，加HIF1α多抗（稀释度1∶200）4℃孵育过夜，TBST漂洗10ｍin×3后，加二抗室温孵育2～3h，洗膜3次，ECL显影。 1.2.2改变细胞内钙对HIF1α转位的影响－