氯化锌对体外培养人晶状体上皮细胞生长的抑制作用及其机制

Suppression and mechanism of zinc chloride on viability of human lens epithelial cells in vitro

背景 锌离子有抑制肿瘤细胞增生、诱导癌细胞凋亡的作用,眼科常用于结膜炎和沙眼治疗的局部用药.迄今为止,关于锌离子对人晶状体上皮细胞（LECs）增生的研究较少.目的 研究氯化锌（ZnCl2）对人LECs细胞株HLEB-3增生的影响及其作用机制.方法 对HLEB-3进行常规细胞培养和传代,在培养基中加入不同质量浓度的ZnCl2,使终质量浓度分别为5、10、20、40和80 mg/L,继续培养细胞24、48和72 h,未添加ZnCl2的细胞作为对照组.采用MTT法检测各组培养细胞的活力;采用JC-1荧光染色法测定细胞线粒体膜电位（△Ψm）的变化;采用流式细胞仪检测不同细胞周期的细胞比例;采用实时荧光定量PCR法和ELISA法分别检测细胞中bcl-2 mRNA及其蛋白的表达量,并对各组检测结果进行比较.结果 对照组培养的细胞呈镶嵌多边形,排列紧密;不同质量浓度ZnCl2组细胞数目均减少,形态不规则,细胞排列紊乱.随着ZnCl2质量浓度的增加和ZnCl2作用时间的延长,细胞生存率均明显降低,总体比较差异均有统计学意义（F质量浓度=173.949,P=0.000;F时间=16.997,P=0.000）.对照组细胞膜呈橙红色荧光,随着ZnCl2质量浓度的增加,不同质量浓度ZnCl2处理后橙红色荧光逐渐减弱,绿色荧光逐渐增强.不同质量浓度ZnCl2组处于S期和G2/M期的细胞比例明显多于对照组,而Go/G1期的细胞明显少于对照组,总体差异均有统计学意义（S期：F=15.594,P=0.000; G2/M期：F=12.792,P=0.000;Go/G1期：F=43.366,P=0.000）.对照组细胞中bcl-2mRNA的相对表达量为1.00±0.00,而5、10、20、40和80 mg/L ZnCl2组bcl-2 mRNA的相对表达量分别降至0.32±0.13、0.07±0.03、0.14±0.05、0.01±0.00和0.12±0.06,差异均有统计学意义（均P＜0.01）;对照组细胞中bcl-2蛋白的表达水平为（138.57±32.80）pg/mg,5、10、20、40和80 mg/L ZnCl2组分别为（79.20±5.70）、（79.95±6.21）、（61.02±10.74）、（72.05±11.61）和（55.97±10.53） pg/mg,均明显低于对照组,差异均有统计学意义（均P＜0.01）.结论 ZnCl2可使HLEB-3细胞阻滞于G2/M期,从而抑制细胞的增生,其作用机制可能与ZnCl2引起△Ψm下降和下调bcl-2在细胞中的表达有关.

Background Zinc ions can inhibit the proliferation of tumor cells and induce the apoptosis of cancer cells and commonly used in the topical treatment of conjunctivitis and trachoma.However,the effect of zinc ions on proliferation of human lens epithelial cells （LECs） is still unclear.Objective The purpose of the present study was to investigate the effect of ZnCl2 on human LECs proliferation and its mechanism.Methods Human LECs strains,HLEB-3 cells,were routinely cultured and passaged.Different concentrations of ZnCl2 was added into the medium to make the final concentrations to 5,10,20,40 and 80 mg/L for 24,48 and 72 hours,and the cultured cells without ZnCl2 were as the control group.Viability of the cells was detected by MTT assay ; the mitochondrial membrane potential （AΨm） was measured using the JC-1 assay kit; the proportion of the cells in different cycles was detected using flow cytometry; meanwhile,the expressions of bcl-2 gene and bcl-2 protein were assayed by real-time quantitative PCR （RT-qPCR） and enzyme-linked immunosorbent assay （ELISA） technique,respectively.Results Cultured cells showed polygon-like with the tight arrangement in the control group.However,the number of the cells was obviously decreased with the irregular shape and disorder arrangement in different concentrations of ZnCl2 groups.The viability of the cells was significantly reduced with the increase of ZnCl2 concentrations and the lapse of active time （F ions =173.949,P =0.000 ; Ftime =16.997,P =0.000）.The strong orange fluorescence in cellular membrane was seen in the control group,but the orange fluorescence was gradually weakened and green fluorescence was gradually enhanced with the increase of concentrations of ZnCl2.The cell proportions in S phase and G2/M phase were significantly raised and those in the Go/G1 phase were declined in different concentrations of ZnCl2 groups in comparison with the control group （S phase：F=15.594,P=0.000; G2/M phase：F=12.792,P=0.000; G0/G1 phase： F=43.366,P =0.000）.The mean relative expression levels of bcl-2 mRNA in the cells were 1.00 ±0.00 in the control group,which were significantly higher than 0.32±0.13,0.07±0.03,0.14±0.05,0.01 ±0.00 and 0.12± 0.06 in the 5,10,20,40 and 80 mg/L ZnCl2 groups （all at P＜0.01）.The expressing levels of bcl-2 protein in the cells were （138.57 ±32.80） pg/mg,which were significantly higher than （79.20±5.70）,（79.95 ±6.21）,（61.02 ±10.74）,（72.05±11.61） and （55.97±10.53） pg/mg in the 5,10,20,40 and 80 mg/L ZnCl2 groups （all at P＜O.01）.Conclusions ZnCl2 can arrest the HLEB-3 cells in G2/M phase and therefore suppresses the proliferation of HLEB-3 cells probably by reducing the AΨm and down-regulating the expression of bcl-2.