阿司匹林诱导人结肠癌细胞Caco-2凋亡与Lgr5的关系  

Effect of Expression of Lgr5 and Apoptosis Induction by Asprin in Colon Cancer Cell Caco-2

作　　者：陈小燕[1] 吴应强[2] 朱蓉[1] 赵逵[1] CHEN Xiaoyan;WU Yingqiang;ZHU Rong;ZHAO Kui(Department of Digestive;Department of Emergency General Medicine,the Affiliated Hospital of Zunyi Medical College,Zunyi 563099,China)

机构地区：[1]遵义医学院附属医院消化内科,遵义563099 [2]遵义医学院附属医院全科医学科,遵义563099

出　　处：《医药导报》2021年第8期1010-1016,共7页Herald of Medicine

基　　金：国家自然科学基金资助项目(81560467);贵州省科学技术基金项目(黔科合LH字[2014]7573号)。

摘　　要：目的研究阿司匹林对人结肠癌细胞株Caco-2的生长作用以及对Lgr5表达的影响,探讨其预防结肠癌发生的可能机制。方法运用噻唑蓝(MTT)比色法检测阿司匹林对Caco-2细胞存活率的影响;流式细胞术分析不同药物浓度和不同时间对Caco-2细胞凋亡的影响;采用实时-反转录聚合酶链反应(RT-PCR)法测定各组阿司匹林干预不同时间后Caco-2细胞中Lgr5 mRNA表达;免疫组化Envison法检测阿司匹林对Lgr5蛋白在Caco-2细胞内定位变化。结果阿司匹林对Caco-2细胞增殖有抑制作用,且有明显的浓度和时间依赖性;对Caco-2细胞促凋亡作用(包括早期凋亡和总凋亡)呈明显的浓度和时间依赖性,不同浓度组和不同时间组与对照组比较均差异有统计学意义(均P<0.05);阿司匹林呈时间依赖性地抑制Caco-2细胞中Lgr5 mRNA的表达,各实验组与对照组比较均差异有统计学意义(均P<0.05);Lgr5蛋白在Caco-2细胞中大量表达,主要位于细胞质,少数定位于细胞膜,而细胞核不着色;5.0 mmol·L^(-1)组中Lgr5蛋白亚细胞定位与对照组相同,10.0 mmol·L^(-1)组和15.0 mmol·L^(-1)组可见Lgr5蛋白亚细胞定位发生了改变,除了细胞膜、细胞质有表达外,细胞核也有表达。结论阿司匹林通过抑制Lgr5基因的表达,并影响Lgr5蛋白亚细胞定位,呈时间和浓度依赖性地抑制体外培养的结肠癌Caco-2细胞的存活以及促进细胞凋亡,可能是阿司匹林防治结直肠癌的重要机制之一。Objective To probe into the possible mechanisms of aspirin for the chemopreventive effects against colon cancer by investigating the effect of aspirin on the growth of human colorectal cancer cells Caco-2 and the expression of Lgr5.Methods MTT assay was used to detect the effect of aspirin on the proliferation of Caco-2 cells;The rate of cell apoptosis was analyzed by flow cytometry;The expression levels of Lgr5 mRNA in Caco-2 cells was detected by real-time reverse-transcription polymerase chain reaction(RT-PCR),respectively.The change of Lgr5 protein localization in Caco-2 cells was detected by immunohistochemistry envison method.Results Aspirin showed time and concentration-dependent antiproliferative effects on the growth of Caco-2 cells.Aspirin at the concentrations of 5.0 mmol·L^(-1),10.0 mmol·L^(-1),15.0 mmol·L^(-1)significantly increased the apoptosis rate in a dose and time-depending manner(P<0.05).The results of RT-PCR showed that aspirin inhibited the mRNA expressions of Lgr5 in a time-dependent manner in Caco-2 cells.There were statistical significances between the experimental groups and the control group(P<0.05).Lgr5 protein was abundantly expressed in Caco-2 cells,which was mainly located in the cytoplasm with a small number located in the cell membrane,while the nucleus was not stained.In the experimental group with 5 mmol·L^(-1)of aspirin,the subcellular localization of Lgr5 protein was the same as that of the control group.the localizations of Lgr5 protein subcells in 10 mmol·L^(-1)group and 15 mmol·L^(-1)group were changed.In addition to cell membrane and cytoplasmic expression,the nucleus was also expressed.Conclusion Aspirin profoundly inhibits Caco-2 cell growth and promotes apoptosis.This effect is associated with reduced mRNA expression of Lgr5 and the affected localization of Lgr5 protein,which may be one of the potential mechanisms of aspirin for the chemoprevention of colorectal cancer.

关 键 词：阿司匹林 结肠癌 CACO-2 LGR5 肿瘤干细胞 

分 类 号：R965[医药卫生—药理学]