三氧化二砷诱导人晶状体上皮细胞凋亡的机制研究

Mechanism of arsenic trioxide induced apoptosis in cultured human lens epithelium cells

目的研究三氧化二砷（As2O3）对离体培养的人晶状体上皮细胞（FHL124）的凋亡及其作用机制。方法实验研究。四甲基偶氮唑盐比色法观察As2O3对FHL124细胞的抑制作用；原位缺13末端标记（TUNEL）法测定细胞凋亡；Taqman实时荧光定量PCR检测基因表达的变化；荧光显微镜动态监测细胞内Cd^2＋浓度的变化。As2O3对FHL124细胞的生长抑制和细胞内Ca^2＋浓度的变化采用t检验进行分析；3种基因不同处理组间表达水平差异的比较采用Wilks’λ检验；单个基因比较采用LSD-t检验。结果As2O3（3×10^-7～1×10^-4mol／L）对FHLl24细胞的作用呈浓度依赖性，1μmol／L As2O3即可显著抑制FHL124细胞的生长，半效抑制量（IC50）为1．5μmol／L。TUNEL检测证实As2O3诱导FHL124细胞凋亡，引起FHL124细胞DNA断裂。实时定量荧光PCR结果显示，As2O3可以引起FHL124细胞与内质网应激信号传导有关的基因产物EIF2A，ERN1和ATF6显著增加（F=8．51，P=0．0005）。细胞内Ca^2＋测定显示As2O3降低细胞内Ca^2＋浓度，从而降低了Ca^2＋信号的峰值。20μmol／L As2O3孵育30min后，峰值降低了（66．34±4．47）％（P=0．0018），60min则降低了（96．95±7．98）％（P=0．0002）。20μmol／L As2O3使Ca^2＋内流幅值及速度分别降低了（22．59±2．98）％和（39．69±6．01）％。结论As2O3抑制FHL124细胞的生长并诱导细胞凋亡，诱导内质网应激可能是其重要作用机制之一。

Objective To study cytotoxic effects of arsenic trioxide （As2O3 ） on the human lens epithelium cells and to identify the biological mechanism for these effects. Methods In this experimental study, human lens epithefium cells （FHL124 cells） were cultured in Eagle＇s minimum essential medium supplemented with 5% fetal calf serum. The effects of As2O3 on FHL124 cells growth were tested by MTT, and apoptosis was detected by TUNEL assay. Gene changes were detected by real-time PCR （Taqman）. As2O3 -induced changes in cell calcium level were measured by real-time fluorometric single-cell digital imaging techniques after Fura-2 incorporation. Results As2O3 inhibited the growth of FHL 124 cells in vitro in a dose-dependent manner, given an IC50 value of 1.5 μmol/L. As2O3 induced apoptosis of FHL124 cells as showed by TUNEL assay. As2O3 provoked an endoplasmic reticulum （ER） stress response identified through an up regulation of EIF2A, ERN1 and ATF6 （ F = 8. 51, P = 0. 0005 ）. As2O3 depleted the calcium store and consequently lead to a decrease of calcium signaling （ P = 0. 0018 ）. Moreover, As2O3 had a moderate effect on the calcium influx pathway. Conclusions As2O3 inhibits the growth and induces apoptosis of human lens epithelium cells. As2O3 provokes an ER stress which could be the cause of apoptic processes.