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目的 探讨细胞分化诱导剂维甲酸对人子宫颈癌细胞系HeLa中缝隙连接蛋白Cx43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 (laserscanningconfocalmicroscope ,LSCM )下 ,动态观察经维甲酸处理后细胞质内信号分子游离钙离子 ( [Ca2 + ]i)浓度及分布变化。应用流式细胞仪 (flowcytometer ,FCM )、结合免疫蛋白电泳(westernblot)分析 ,检测外源信号分子对Cx43蛋白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 ,[Ca2 + ]i由静息状态下的 3 5 .73 μmol/L上升至 5 8.16μmol/L。流式细胞仪分析 ,Cx43蛋白阳性细胞计数率由1.9%上升至 2 6.3 %。Westernblot分析HeLa细胞出现Cx43蛋白酪氨酸磷酸化。结论 HeLa细胞Cx43信号转导途径是在外源信号刺激下 ,在细胞质内 [Ca2 + ]i的参与下 ,Cx43蛋白表达上调 ,并在酪氨酸位点出现明显的磷酸化 ,维甲酸通过对与生长抑制相关的cx基因信号转导途径的调节 ,实现对肿瘤生长的抑制作用。
Objective To investigate the regulation of connexin Cx43 signal transduction pathway in cervical cancer cell line HeLa by cell differentiation inducer Retinoic acid. Methods The concentration and distribution of free calcium ions ([Ca2 + ]i) in the cytoplasm of retinoid acid were dynamically observed under a laser scanning confocal microscope (LSCM) using the specific calcium indicator Fluo 3AM. Flow cytometer (FCM) and western blot analysis were used to detect the effect of exogenous signal molecules on the expression of Cx43 protein and protein tyrosine phosphorylation status. Results The free calcium in HeLa cells was significantly overloaded by retinoic acid and [Ca2 +]i increased from 33.73 μmol/L to 5 8.16 μmol/L in resting state. Flow cytometry analysis showed that the positive rate of Cx43 protein increased from 1.9% to 26.3%. Western blot analysis of tyrosine phosphorylation of Cx43 protein in HeLa cells. Conclusion The Cx43 signal transduction pathway of HeLa cells is stimulated by exogenous signal, and the expression of Cx43 protein is up-regulated in the presence of [Ca2 +]i in the cytoplasm, and phosphorylation is apparent at the tyrosine locus. Inhibition of growth-associated cx gene signal transduction pathways to achieve inhibition of tumor growth.