过表达calbindin-d28k在卡铂诱导子宫内膜癌细胞凋亡中的作用及机制

Effects of Calbindin-d28k Over-expression on Carboplatin-induced Apoptosis in Human Endometrial Cancer Cells and Its Mechanism

[目的]研究过表达钙结合蛋白calbindin-d28k对卡铂诱导子宫内膜癌Ishikawa细胞凋亡的影响及其可能的作用机制。[方法]构建calbindin-d28k基因真核过表达calbindind28k质粒,稳定转染子宫内膜癌Ishikawa细胞;实时荧光定量PCR（q RT-PCR）及免疫印迹方法检测细胞中靶基因m RNA及蛋白的表达;MTT法检测不同浓度卡铂作用下子宫内膜癌Ishikawa细胞的存活率Hoechst 33258染色法观察卡铂诱导的子宫内膜癌Ishikawa细胞胞核变化。[结果]卡铂对子宫内膜癌Ishikawa细胞的最佳作用浓度为100μg/ml,可显著性上调细胞活性Caspase-3蛋白的表达（P〈0.05）;稳定转染组Ishikawa细胞calbindin-d28k m RNA水平上调约10倍,蛋白水平升高约1.6倍（P〈0.05）;过表达calbindin-d28k后,卡铂诱导子宫内膜癌Ishikawa细胞的凋亡作用减弱,活性Caspase-3蛋白的表达水平下调约7倍（P〈0.05）。[结论 ]过表达calbindin-d28k蛋白可抑制卡铂诱导的子宫内膜癌Ishikawa细胞的凋亡作用,并与通过下调Caspase-3有关。

[Objective] To investigate the effect of calbindin-d28k over-expression on carboplatin- induced apoptosis in endometrial cancer cells and the related mechanism. [ Methods] A eukaryot-ic expression vector of human calbindin-d28k gene was constructed and transfected into human endometrial cancer Ishikawa cells. RT-PCR and western blotting were used to analyze the mRNA and protein expression of calbindin-d28k genes in Ishikawa cells,respectively. The Ishikawa cells were treated with different concentrations of carboplatln and the cell survival rate in were observed by MTT assay. Hoechst 33258 assay was used to detect the nuclear variation of Ishikawa cells induced by carboplatin.[Results] The levels of mRNA and protein expression of calbindin- d28k gene in the stable transfected Ishikawa cells were 10 and 1.6 times higher than those in vector group,respectively. The optimal concentration of carboplatin was 100μg/ml in Ishikawa cells, at which the expression of cleaved Caspase-3 was significantly increased. The cell apoptosis and Caspase-3 expression level were reduced by 7 times in Ishikawa cells with over-expression of calbindin-d28k. [Conclusion] The over-expression of calbindin-d28k inhibits carboplatin-induced apoptosis in endometrial cancer,which is related to the decreased expression of Caspase-3.