维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白转导途径调控作用的研究

Phosphorylation of gap junction gene connexin 43 protein and dissociated calcium in HeLa cell line by all-trans -retinoic acid

目的 探讨维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白 (Cx) 4 3信号转导途径的调控作用。方法 应用特异性钙指示剂 (Fluo 3AM)在激光扫描共聚焦显微镜下动态观察维甲酸作用后的细胞质内信号转导分子游离钙的分布及强度变化。采用流式细胞仪、结合蛋白印迹技术分析 ,检测外源信号分子对Cx43的表达以及蛋白酪氨酸磷酸化状态的影响。结果 HeLa细胞内游离钙经维甲酸作用后明显超载 ,细胞内游离钙浓度 ([Ca2 + ]i)由静息状态下的 35 73μmol/L上升至5 8 16 μmol/L。流式细胞仪分析 ,Cx43阳性细胞表达率由 1 9%上升至 2 6 3%。蛋白印迹技术分析 ,HeLa细胞出现Cx43酪氨酸磷酸化。结论 维甲酸对HeLa细胞Cx43信号转导途径的调控是在细胞质内游离钙的参与下 ,使Cx43阳性细胞表达率上升 。

Objective To investage the regulation effect on signal transduction pathway of gap junction gene connexin (Cx)43 in human cervical carcinoma cell line HeLa by all trans retinoic acid(ATRA) Methods Cell culture, Fluo 3 AM loading and laser scanning confocal microscope, flowcytometer(FCM)and western blot were employed to detect expression and regulation effect on signal transduction pathway of gap junction gene connexin Cx43 in HeLa cells by retinotic acid(RA) Results After treated with ATRA, the intercellular second messenger dissociated calcium ([Ca 2+ ]i) was much higher in HeLa cells (58 16 μmol/L)than untreated cell(35 73 μmol/L) A detectable and up regulation of Cx43 of 43 000 protein in HeLa cells inreased from 1 9% to 26 3% in RA treated cells than untreated cell examined by FCM and western blot Immunoblot analysis showed that only the treated cells had phosphorylated form of Cx43 protein Conclusion The anti tumor effect of ATRA in HeLa cells might be due to up regulation of Cx43 gene and its signal transduction pathway which mediats gap junction intercellular communication