深低温暴露诱导的人肾小管上皮细胞自噬体数量特征分析  

Quantitative Characteristics of Autophagosomes in Human Renal Tubular Epithelial Cells Induced by Deep Hypothermic Exposure

作　　者：杨希营 王古岩[1,2] 陈一萌 张丛雅 杜英杰 YANG Xi-ying;WANG Gu-yan;CHEN Yi-meng;ZHANG Cong-ya;DU Ying-jie(Department of Anesthesiology,Fuwai Hospital,National Center for Cardiovascular Disease,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100037,China;Department of Anesthesiology,Beijing Tongren Hospital,Capital Medical University,Beijing 100730,China)

机构地区：[1]北京协和医学院国家心血管病中心中国医学科学院阜外医院麻醉科,北京市100037 [2]首都医科大学附属北京同仁医院麻醉科,北京市100730

出　　处：《中国分子心脏病学杂志》2021年第1期3744-3747,共4页Molecular Cardiology of China

基　　金：国家自然科学基金(81770414,81970344)。

摘　　要：目的通过构建HK2-GFP-LC3稳定细胞系,研究20℃深低温暴露诱导的自噬体数量特征,为明确自噬低温暴露响应和自噬相关药物高通量筛选提供依据。方法构建稳定表达GFP-LC3融合蛋白的HK2-GFP-LC3细胞系,建立HK2-GFP-LC3细胞体外20℃低温暴露自噬分析模型,用荧光显微镜进行自噬体计数,进行低温暴露和自噬体数量相关性分析,建立自噬体数量和低温暴露时长的数学回归模型。结果HK2-GFP-LC3细胞GFP-LC3融合蛋白阳性表达率达到100%,指示自噬体的绿色明亮荧光斑点(GFP-LC3 spots)数量在20℃暴露后随时间逐渐增加,GFP-LC3 spots阳性细胞率和低温暴露时长(h)呈直线正相关,拟合公式为Y=1.649×X+17.41(解释为暴露时间每增加1h,细胞GFP-LC3 spots阳性率增加1.649%)。复温后GFP-LC3 spots数量逐渐下降。GFP-LC3 spots数量在复温前后表现为倒U形曲线发展变化过程。结论体外低温暴露能够时间依赖性激活细胞自噬,复温可能能够抑制自噬。Objective To study the quantitative characteristics of autophagosomes induced by deep hypothermic exposure at 20℃in constructed HK2-GFP-LC3 stable cell lines for the identification of autophagy and the high-throughput screening of autophagy-related agents.Methods HK2-GFP-LC3 cell lines with stable expression of GFP-LC3 fusion protein were constructed to establish cold exposure model at 20Ⅱin vitro for autophagosome analysis.The autophagosomes were counted by fluorescence microscope and the correlation analysis between hypothermic exposure and autophagosomes was conducted subsequently.Meanwhile a linear regression model was established.Results GFP-LC3 spots increased gradually after exposure at 20℃.There was a linear positive correlation between the number of GFP-LC3 spots and the duration of hypothermic exposure,and the fitting formula was Y=1.649×X+17.41.GFP-LC3 spots decreased gradually after rewarming.The trendline of GFP-LC3 spots showed an inverted U-shaped curve in the process of hypothermic exposure and rewarming.Conclusion Hypothermic exposure can activate autophagy in a time-dependent manner in vitro.Rewarming can likely inhibit autophagy.

关 键 词：自噬 自噬体 低温暴露 急性肾损伤 

分 类 号：R318.52[医药卫生—生物医学工程]