【摘要】 目的 探讨十字花科蔬菜的天然植物化合物3,3′-二吲哚甲烷(3,3′-diindolelmethane, DIM)在胃癌防治中的作用及其分子机制，为膳食植物营养应用于肿瘤预防提供实验依据。方法 采用不同浓度的DIM(0、20、40、60、80、100、120μmol/L)处理人胃癌细胞系BGC-823 24 h后，采用四甲基偶氮唑蓝(MTT)法测定各组细胞存活率，Western blot检测各组细胞钙蛋白酶(Calpain)、激活转录因子4(ATF4)和CAAT/增强子结合蛋白同源蛋白(CHOP)蛋白的表达水平；钙离子荧光探针检测细胞内钙离子浓度变化；使用激活转录因子4(ATF4)的小干扰RNA(siRNA)特异性敲减胃癌细胞系中的ATF4,并用Western blot检测ATF4和CHOP蛋白的表达水平，MTT法检测细胞存活率；应用钙离子螯合剂BAPTA-AM预处理胃癌细胞系BGC-823后，Western blot检测Calpain、ATF4和CHOP蛋白的表达水平，MTT法检测细胞增殖活力改变情况。结果 MTT法、Western blot及钙离子荧光探针法检测结果表明，随着DIM浓度升高，BGC-823细胞的增殖能力显著降低(P<0.05),Calpain、ATF4和CHOP蛋白的表达上调(P<0.05),细胞内钙离子荧光强度增强；转染ATF4 siRNA后，BGC-823细胞ATF4蛋白的表达降低(F=16.17,P<0.05),且与DIM+si-NC组相比，DIM+si-ATF4组细胞存活率增加(F=74.46,P<0.05);进一步通过BAPTA-AM螯合细胞内钙离子后，与DIM组相比，DIM+BAPTA-AM组Calpain、ATF4和CHOP蛋白的表达降低(F分别为24.58、34.93、24.45,P<0.05),细胞存活率增加(F=31.13,P<0.05)。结论 DIM可增加细胞质钙离子浓度，上调Calpain、ATF4和CHOP蛋白的表达水平，从而诱导胃癌细胞系发生内质网应激，抑制胃癌细胞的增殖。更多还原

【Abstract】 Objective To investigate the role and molecular mechanism of 3,3′-diindolelmethane(DIM), a natural plant compound in cruciferous vegetables, in the prevention and treatment of gastric cancer, and then provide experimental evidence for the application of dietary plant nutrition in the prevention of gastric cancer. Methods After human gastric cancer cell line BGC-823 was treated with different concentrations of DIM, including 0, 20, 40, 60, 80, 100, and 120 μmol/L, for 24 hours, the cell survival rate was determined by the tetramethylazolium blue(MTT) method. Western blot was used to detect the expression levels of Calpain, activating transcription factor 4(ATF4) and CAAT/enhancer-binding protein homologous protein(CHOP). The changes of intracellular calcium ion concentration were detected by a calcium ion fluorescent probe. After ATF4 in BGC-823 cells was specifically knocked down using the small interfering RNA(siRNA) of ATF4, the expression levels of ATF4 and CHOP were detected by Western blot and the cell survival rate was determined by the MTT method. After the BGC-823 cells were pretreated with the calcium ion chelator BAPTA-AM, the expression levels of calpain, ATF4 and CHOP proteins were measured by Western blot and the changes in cell proliferation activity were determined by the MTT method. Results The detection results of the MTT, Western blot, and calcium ion fluorescent probe methods showed that with the increase of DIM concentration, the proliferation ability of BGC-823 cells significantly decreased(P<0.05). Meanwhile, the expressions of Calpain, ATF4, and CHOP proteins were up-regulated(P<0.05) and the fluorescence intensity of intracellular calcium ion increased. After transfection with the siRNA of ATF4, the expression level of ATF4 in BGC-823 cells was decreased(F=16.17, P<0.05). Compared with the DIM+si-NC group, the cell survival rate in the DIM+si-ATF4 group was increased(F=74.46, P<0.05). After the intracellular calcium ions were chelated by BAPTA-AM, the expression levels of Calpain, ATF4, and CHOP proteins in the DIM+BAPTA-AM group were significantly lower than that in the DIM group(F=24.58, 34.93, and 24.45, respectively, P<0.05), while the cell survival rate was the opposite(F=31.13, P<0.05). Conclusion DIM can increase the concentration of intracellular calcium ions and up-regulate the expressions of Calpain, ATF4 and CHOP proteins, thereby inducing the endoplasmic reticulum stress in gastric cancer cells and inhibiting their proliferation.更多还原