摘要
目的维生素E琥珀酸酯(vitamin E sucinate,VES)是天然维生素E衍生物中抑制肿瘤活性最强的一种,国内外研究已证实VES在体内外均能有效抑制肿瘤细胞生长,而对正常细胞无任何毒副作用。本研究旨在探讨VES是否通过肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)途径诱导胃癌细胞凋亡,及VES是否可促进人CD4^+T淋巴细胞表达TRAIL及其受体,并探讨在体外联合应用VES与CD4^+T淋巴细胞是否可增强对人胃癌细胞的凋亡诱导作用。方法以中分化人胃癌MKN28细胞作为体外肿瘤模型。体外培养人胃癌MKN28细胞,用20μg/mL VES处理胃癌细胞不同时间(0、6、12、18和24 h)后,流式细胞仪检测各组凋亡率;不同剂量(0、5、10和20μg/mL)VES处理MKN28细胞12 h,或10μg/mL VES分别处理MKN28细胞不同时间(0~24 h)后,利用RT-PCR与蛋白质印迹法分别检测细胞内TRAIL受体DR4、DR5的mRNA和蛋白表达水平;用特异siRNA分别沉默人胃癌MKN28细胞DR4、DR5表达后,加入20μg/mL VES处理细胞24 h,流式细胞仪检测各组凋亡率;不同剂量(0、5、10和20μg/mL)VES处理人CD4^+T细胞12 h后,利用RT-PCR分析人CD4^+T细胞中TRAIL和TRAIL受体的mRNA表达情况;将CD4^+T细胞与胃癌细胞共培养后用20μg/mL VES处理,将实验设置为对照组(Control)、CD4^+T细胞作用组、VES作用组和CD4^+T细胞+VES联合作用组,光镜下观察细胞形态变化;并进行DAPI染色,荧光显微镜观察细胞核与染色质形态变化。结果20μg/mL VES处理胃癌细胞0、6、12、18和24 h,各组凋亡率分别为(2.8±0.46)%、(4.37±0.06)%、(7.27±0.64)%、(29.7±4.17)%和(42.02±2.35)%,不同作用时间组凋亡率之间差异有统计学意义,F=198.288,P<0.001;RT-PCR及蛋白质印迹法结果显示,10μg/mL VES作用下,DR4、DR5的mRNA和蛋白表达均呈先升高后降低的趋势,DR4、DR5的mRNA分别在9和6 h处达到峰值,而蛋白表达在12 h达到峰值;用不同剂量VE
OBJECTIVE Vitamin E succinate(VES)is the strongest one to inhibit tumer activity in natural vitamin E derivatives,research at home and abroad have proved that VES can effectively inhibit the growth of tumor cells in vivo and in vitro without any toxic and side effects on normal cells.This study continued to explore whether VES can induce apoptosis of gastric cancer cells through TRAIL pathway,and whether VES can promote the expression of TRAIL and its receptor in human CD4^+T lymphocytes,and whether the combined application of VES and CD4^+T lymphocytes in vitro can enhance the apoptosis induction effect of human gastric cancer cells.MEDHODS MKN28 cells of monderately differentiated human gastric cancer were used as in vitro tumor medel.Human gastric cancer MKN28 cells were cultured in vitro and treated with 20μg/mL VES for different time(0,6,12,18,24 h).The apoptosis rate of each group was detected by FACS.After MKN28 cells were treated by VES for 12 hat different doses(0,5,10,20μg/mL),or MKN28 cells were treated by 10μg/mL VES for different times(0-24 h),RT-PCR and western blot were used to respectively detecte mRNA and protein expression levels of TRAIL receptor DR4 and DR5.Specific siRNA was used to silence the expression of DR4 and DR5 in human gastric cancer MKN28 cells,and the cells were treated with 20μg/mL VES for 24 h.The apoptosis rate of each group was detected by FACS.After treating human CD4^+T cells with different doses(0,5,10,20μg/mL)for12 h,TRAIL and TRAIL receptor mRNA expression in human CD4^+T cells were analyzed by RT-PCR.CD4^+T cells were co-cultured with gastric cancer cells and treated with 20μg/mL VES.The experimental group was set as the control group,CD4^+T action group,VES action group and CD4^+T cells+VES combined action group.Changes of cell morphology were observed under light microscope,and staining the cells with the DAPI,fluorescence microscope was used to observe the morphological change of nucleus and chromatin.RESULTS After 20μg/mL VES treated gastric cancer cells for0,
作者
苑瑾慧
杜美志
王奕丹
侯丽颖
YUAN Jin-hui;DU Mei-zhi;WANG Yi-dan;HOU Li-ying(School of Public Health,North China University of Science and Technology,Tangshan 063020,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2020年第8期596-604,共9页
Chinese Journal of Cancer Prevention and Treatment
基金
青年科学基金(H2019209453)。
