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。

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

温晶, 常晓岑, 郭丹, 都健

中国医科大学附属第四医院内分泌代谢内科, 沈阳 110032

收稿日期:2021-04-14出版日期:2022-02-28发布日期:2022-01-05
通讯作者:都健E-mail:dujianbox@126.com
作者简介:温晶(1987-),女,主治医师,博士研究生.
基金资助: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）


关键词: 增食欲素A, 自噬, 凋亡, HT-29细胞
Abstract: 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 admini-stration. 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.
Key words: Orexin A, autophagy, apoptosis, HT-29 cells
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