论文部分内容阅读
目的探讨PTEN基因是否增强子宫内膜癌细胞系Ishikawa细胞对阿霉素的敏感性。方法将PTEN基因转染前、后的Ishikawa细胞分别暴露于系列浓度的阿霉素,以MTT法测定这些细胞对阿霉素的敏感性,并通过Hoechst33258染色荧光显微镜观察细胞凋亡情况。同时,以免疫沉淀Westernblot法观察阿霉素对Bad和Akt/PKB蛋白磷酸化的影响。结果阿霉素以剂量依赖方式诱导全部细胞系的细胞凋亡,但对表达PTEN的克隆细胞所诱导的凋亡比对亲本株Ishikawa细胞更明显。低浓度阿霉素(0.1μmol/L)不引起PTEN蛋白缺失的Ishikawa细胞凋亡,但却能诱导表达PTEN蛋白克隆细胞凋亡;高浓度阿霉素(1μmol/L)诱导全部细胞系的细胞凋亡,但凋亡的发生率在表达PTEN的克隆细胞中明显高于亲本株Ishikawa细胞。在表达PTEN的克隆细胞中,磷酸化Akt/PKB和磷酸化Bad(Ser-136)的水平均下降。阿霉素降低了全部细胞系的磷酸化Akt/PKB和磷酸化Bad(Ser-136)水平,但在表达PTEN的克隆细胞中降低最明显。结论PTEN基因转染明显地增强了Ishikawa细胞对阿霉素的化学敏感性,阿霉素可能是通过下调PI3k/Akt/PKB信号途径而与PTEN蛋白共同发挥凋亡诱导作用。
Objective To investigate whether PTEN gene enhances the sensitivity of doxorubicin to Ishikawa cells in endometrial carcinoma cell line. Methods Ishikawa cells before and after transfection of PTEN gene were respectively exposed to a series of doxorubicin concentrations. The sensitivity of these cells to doxorubicin was determined by MTT assay. Cell apoptosis was observed by Hoechst 33258 staining. At the same time, immunoprecipitation Western blot was used to observe the effects of doxorubicin on the phosphorylation of Bad and Akt / PKB proteins. Results Doxorubicin induces apoptosis in all cell lines in a dose-dependent manner but induces apoptosis more strongly in PTEN-expressing clonal cells than in parental Ishikawa cells. Low concentrations of doxorubicin (0.1 μmol / L) did not induce PTEN protein-deficient Ishikawa cells apoptosis but induced apoptosis of PTEN-expressing cloned cells; high concentrations of doxorubicin (1 μmol / L) induced cells in all cell lines However, the incidence of apoptosis in PTEN-expressing clonal cells was significantly higher than that of the parental strain Ishikawa cells. In PTEN-expressing clones, the levels of phosphorylated Akt / PKB and phosphorylated Bad (Ser-136) all decreased. Adriamycin decreased the levels of phosphorylated Akt / PKB and phosphorylated Bad (Ser-136) in all cell lines but was most pronouncedly decreased in PTEN-expressing clonal cells. Conclusion PTEN gene transfection significantly enhances chemosensitivity of doxorubicin to Ishikawa cells. Adriamycin may play an apoptosis inducing role with PTEN protein through down-regulating PI3k / Akt / PKB signaling pathway.