增食欲素A诱导的自噬在人结肠癌细胞HT-29凋亡中的保护作用  

Protection of HT-29 human colon cancer cells from apoptosis by Orexin A-induced autophagy

作　　者：温晶 常晓岑 郭丹[1] 都健[1] WEN Jing;CHANG Xiaocen;GUO Dan;DU Jian(Department of Endocrinology and Metabolism,The Fourth Affiliated Hospital of China Medical University,Shenyang 110032,China)

机构地区：[1]中国医科大学附属第四医院内分泌代谢内科,沈阳110032

出　　处：《中国医科大学学报》2022年第2期140-144,共5页Journal of China Medical University

基　　金：辽宁省博士科研启动基金(2019-BS-298);中国医科大学2018年青年骨干支持计划(QGZD2018070)。

摘　　要：目的研究增食欲素A(Orexin A)诱导的人结肠癌细胞HT-29自噬性活化,探讨自噬与凋亡的关系。方法用Orexin A处理培养的HT-29细胞,通过透射电子显微镜观察细胞内自噬体的形成情况。用不同浓度Orexin A处理HT-29细胞,通过Western blotting测定自噬相关蛋白Beclin-1和LC3的表达水平。用氯喹(CQ)和Orexin A共同处理细胞,通过MTT法检测对细胞活力的影响,用流式细胞仪检测细胞的凋亡情况,Western blotting检测caspase-3的裂解。结果Orexin A处理后,HT-29细胞活力降低,凋亡率升高,电镜下可见细胞内自噬体形成,Beclin-1和LC3蛋白的表达水平均升高。与单独Orexin A作用相比,CQ诱导细胞的凋亡率增加,增殖抑制率降低,Orexin A可裂解前caspase-3至其活性形式。结论Orexin A能够诱导HT-29细胞发生自噬性活化,延缓凋亡的发生。Objective To study the activation of autophagy in HT-29 human colon cancer cells by Orexin A,and to explore relationships between autophagy and apoptosis.Methods HT-29 cells were cultured in vitro and divided into a control group,and an Orexin A-treated group.Autophagosomes were observed using transmission electron microscopy.Expression levels of autophagy-related proteins Beclin-1 and LC3 were determined by Western blotting.Autophagy was down-regulated by chloroquine(CQ).The effect of Orexin A on HT-29 cell viability was determined by MTT assay,and apoptotic cells were detected by flow cytometry.Caspase-3 cleavage was observed and quantitated by Western blotting.Results HT-29 cell activity was decreased,and the apoptosis rate was increased after treatment with Orexin A.Autophagosomes were visible by electron microscopy.Beclin-1 and LC3 protein expression levels were increased after Orexin A administration.Compared to Orexin A treatment alone,HT-29 cell apoptosis was increased,and inhibition of cell proliferation was decreased by CQ pretreatment.Orexin A cleaved pro-caspase 3 to its active form.Conclusion Orexin A can induce protective autophagy and prevent apoptosis of HT-29 cells.

关 键 词：增食欲素A 自噬 凋亡 HT-29细胞 

分 类 号：R735.35[医药卫生—肿瘤]