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目的 :探讨稳定表达电压门控氯通道3(voltage-gated chloride channel 3,ClC-3)的宫颈癌HeLa细胞对顺铂敏感性的变化及其可能的机制。方法 :通过脂质体介导的方法将重组载体质粒pcDNA3.1/ClC-3转染至HeLa细胞中。pcDNA3.1/ClC-3转染后,蛋白质印迹法检测细胞中ClC-3蛋白和P-糖蛋白(P-glycoprotein,P-gp)的表达水平,实时荧光定量PCR法检测细胞中ClC-3和多药耐药基因1(multi-drug resistance gene 1,MDR1)mRNA的表达水平,MTT法检测顺铂对细胞增殖的影响,免疫荧光法检测细胞中ClC-3蛋白和P-gp的表达及定位。结果 :成功获得稳定表达ClC-3的HeLa/ClC-3细胞。HeLa/ClC-3细胞中ClC-3蛋白和P-gp的表达水平以及MDR1和ClC-3 mRNA的表达水平均明显高于转染空载体pcDNA3.1的HeLa/mock细胞,差异有统计学意义(P<0.05)。与转染空载体pcDNA3.1的HeLa/mock细胞比较,顺铂对HeLa/ClC-3细胞增殖的抑制作用明显降低,差异有统计学意义(P<0.05),顺铂对HeLa/ClC-3细胞的半数抑制浓度(half inhititory concentration,IC50)为71μmol/L,明显高于HeLa/mock细胞的32μmol/L(P<0.05)。ClC-3蛋白和P-gp共同定位于HeLa/ClC-3和HeLa/mock细胞的细胞膜。结论 :ClC-3可降低HeLa细胞对顺铂的敏感性,可能与ClC-3上调P-gp的表达水平有关。
Objective: To investigate the changes of sensitivity to cisplatin and its possible mechanism in HeLa cells stably expressing voltage-gated chloride channel 3 (ClC-3). Methods: The recombinant plasmid pcDNA3.1 / ClC-3 was transfected into HeLa cells by liposome-mediated method. After transfection with pcDNA3.1 / ClC-3, the expression of ClC-3 protein and P-glycoprotein (P-gp) were detected by Western blotting. The expression of ClC-3 And multidrug resistance gene 1 (MDR1) mRNA expression levels were detected by MTT assay of cell proliferation, the expression of ClC-3 protein and P-gp in cells were detected by immunofluorescence Positioning. Results: HeLa / ClC-3 cells stably expressing ClC-3 were successfully obtained. The expression of ClC-3 protein and P-gp in HeLa / ClC-3 cells and the expression levels of MDR1 and ClC-3 mRNA in HeLa / ClC-3 cells were significantly higher than those in HeLa / mock cells transfected with empty vector pcDNA3.1 (P <0.05). Compared with HeLa / mock cells transfected with empty vector pcDNA3.1, the inhibitory effect of cisplatin on HeLa / ClC-3 cell proliferation was significantly decreased (P <0.05), and the effect of cisplatin on HeLa / ClC-3 The cell half-concentration concentration (IC50) was 71μmol / L, which was significantly higher than that of HeLa / mock cells (32μmol / L) (P <0.05). ClC-3 protein and P-gp co-localized in the cell membrane of HeLa / ClC-3 and HeLa / mock cells. Conclusion: ClC-3 can reduce the sensitivity of HeLa cells to cisplatin, which may be related to the up-regulation of P-gp expression by ClC-3.