ClC-3氯离子通道对高分化鼻咽癌细胞突起形成的影响

Effect of ClC-3 chloride channel on protrusion formation in well-differentiated nasopharyngeal carcinoma cells

目的:探讨ClC-3氯离子通道在高分化鼻咽癌CNE-1细胞突起形成中的作用。方法:采用免疫荧光技术(Alexa Fluor 488标记抗体)检测CNE-1细胞中ClC-3蛋白的分布;MQAE荧光探针检测细胞内氯离子浓度;吸附式单通道膜片钳技术记录氯离子单通道活动;采用FAM标记的siRNA转染细胞,实验分为对照组、阴性对照(NC) siRNA组和ClC-3 siRNA组;激光共聚焦显微镜观察转染后绿色荧光情况,流式细胞术检测转染效率,Western blot法测ClC-3蛋白的表达,倒置显微镜下观察CNE-1细胞突起形成情况。结果:ClC-3蛋白主要分布于细胞膜和细胞质,且在细胞膜突起形成部位明显聚集;细胞突起处MQAE荧光弱,氯离子浓度较高,而突起根部荧光强,氯离子浓度较低;突起根部记录到翻转电位为4.06 mV的单通道电流,在-120 mV钳制电压下,该通道电导约为78.4pS;siRNA转染细胞48 h后,ClC-3 siRNA组细胞ClC-3蛋白表达量显著降低(P<0.01),且ClC-3 siRNA组细胞无片状伪足伸出,而对照组和NC siRNA组细胞伸出明显的片状伪足;此外,3组细胞均有数量不等的丝状伪足形成。结论:ClC-3影响CNE-1细胞贴壁生长时胞膜伸出突起、形成片状伪足的过程。

AIM:To investigate the role of ClC-3 chloride channel in the protrusion formation of well-differentiated nasopharyngeal carcinoma CNE-1 cells. METHODS:The distribution of ClC-3 protein in CNE-1 cells was explored via immunofluorescence with Alexa Fluor 488 labeled antibody. The intracellular chloride ions were detected by MQAE fluorescence probe. The activity of the single chloride channel was recorded with the single-channel patch-clamp of the cell-attached model. The cells were transfected with the FAM-labeled siRNA, and were divided into control group, negative control(NC) siRNA group and ClC-3 siRNA group. The green fluorescence of the transfected cells, the transfection efficiency of siRNA and the expression of ClC-3 protein were assayed by laser confocal microscopy, flow cytometry, and Western blot, respectively. The protrusion formation of CNE-1 cells in the 3 groups were observed under inverted microscopy. RESULTS:The ClC-3 protein was primarily localized in the cell membrane and plasma, and obviously clustered in the formation sites of membrane protrusions. There were low MQAE fluorescence and high Cl-concentration in the cell extension, while high MQAE fluorescence and low Cl-concentration were observed in the root of the cell extension. The current of single ion channel was recorded in the root of the cell extension, the reversal potential was 4. 06 mV, and the conductivity was 78. 4 pS at-120 mV. The ClC-3 protein was significantly decreased in CNE-1 cells after transfected with ClC-3 siRNA for 48 h(P<0. 01). The cells in control and NC siRNA groups extended lamellipodia, while the cells in ClC-3 siRNA group had no extensions of the lamellipodia. In addition, the cells in the 3 groups formed filopodia in varied numbers. CONCLUSION:The ClC-3 protein affects the extension of the cell membrane to form the lamellipodia when nasopharyngeal carcinoma CNE-1 cells grow on the matrix.