低压低氧对肠黏膜屏障及LINE-1核酸内切酶变异体GCRG213表达的影响  

Effects of hypobaric and hypoxia on barrier function of intestinal mucosa and expression of LINE-1 endonuclease variant GCRG213

作　　者：张王静怡 张少杰 陶虹锦 孟繁森 刘静[3] 朱玲玲 王刚石[2] ZHANG Wangjingyi;ZHANG Shaojie;TAO Hongjin;MENG Fansen;LIU Jing;ZHU Lingling;WANG Gangshi(Chinese PLA Medical School,Beijing 100853,China;Department of Gastroenterology,the Second Medical Center&National Clinical Research Center for Geriatric Diseases,Chinese PLA General Hospital,Beijing 100853,China;Institute of Geriatrics,the Second Medical Center,Chinese PLA General Hospital,National Clinical Research Center for Geriatrics Diseases,Beijing 100853,China;Institute of Military Cognition and Brain Sciences,Beijing 100850,China)

机构地区：[1]解放军医学院,北京100853 [2]解放军总医院第二医学中心消化科,国家老年疾病临床医学研究中心,北京100853 [3]解放军总医院第二医学中心老年医学研究所,衰老及相关疾病研究北京市重点实验室,北京100853 [4]军事认知与脑科学研究所,北京100850

出　　处：《解放军医学院学报》2024年第3期296-301,F0003,共7页Academic Journal of Chinese PLA Medical School

摘　　要：背景长散在核元件-1(long interspersed nuclear element-1,LINE-1或L1)是目前基因组中唯一活跃的自主反转录转座元件,低压低氧环境可以改变L1的甲基化程度。目的探究低压低氧环境对肠黏膜中L1核酸内切酶(L1 endonuclease,L1-EN)变异体GCRG213表达水平及以闭合蛋白(Occludin)和紧密连接蛋白-1(Claudin-1)表达水平为指标的肠黏膜机械屏障功能的影响。方法利用免疫组织化学染色(immunohistochemical staining,IHC)检测人正常小肠、结肠组织及小鼠正常结肠组织的GCRG213表达,观察GCRG213蛋白在正常肠道中的分布规律。将雄性C57BL/6小鼠随机分为实验组和对照组。实验组模拟海拔6000 m环境,饲养7 d构建低压低氧小鼠模型,对照组常压常氧饲养。将人正常结肠上皮细胞NCM-460随机分为常氧组以及低氧24 h、48 h组,常氧组于常氧环境正常培养,低氧组分别于0.3%O2浓度下培养24 h、48 h构建低氧细胞模型。使用qPCR、Western blot技术检测GCRG213、Occludin和Claudin-1在小鼠结肠组织及人NCM-460细胞中表达水平的变化。结果人正常小肠、结肠及小鼠结肠组织IHC结果一致显示,GCRG213表达于肠黏膜柱状上皮细胞胞质中,而在杯状细胞中未见表达。在动物实验中,与常氧组相比,低压低氧组小鼠结肠组织GCRG213在mRNA水平(P<0.05)和蛋白水平(P<0.01)均表达上调;同时,Occludin在蛋白水平表达下调(P<0.05)。在细胞实验中,低氧暴露后NCM-460细胞中GCRG213在mRNA(P<0.001)和蛋白水平(P<0.01)均表达上调,其中低氧24 h组GCRG213表达水平低于低氧48 h组,Occludin、Claudin-1在mRNA水平(P<0.01;P<0.001)表达下调,其中低氧24 h组表达水平均低于低氧48 h组,Occludin蛋白水平表达在低氧48 h组出现下调(P<0.05)。结论L1-EN变异体GCRG213在人和小鼠正常结肠组织黏膜柱状上皮细胞胞质内存在表达;在体内外模型中,低压低氧暴露下结肠黏膜上皮细胞GCRG213表达升高,同时观察到低压Background Long interspersed nuclear element-1(LINE-1 or L1)is currently the only active autonomous retrotransposon in the genome,and its activity is suppressed by high levels of methylation.Hypobaric and hypoxia environments can affect the methylation level of L1.Objective To investigate the impact of hypobaric and hypoxia environments on the mechanical barrier function of intestinal mucosa as measured by expression levels of L1 endonuclease variants GCRG213,Occludin,and Claudin-1.Methods The expression of GCRG213 in normal human small intestine,colon and mouse colon tissues were detected by immunohistochemical staining(IHC)to observe the distribution pattern of the GCRG213 protein in the intestines.Male C57BL/6 mice were randomly divided into control group and experimental group.The hypobaric hypoxia mouse model was established by exposing them to a simulated high-altitude environment of 6000m for 7 days.NCM-460 cells derived from human normal colon epithelial cells were randomly divided into normal oxygen group and hypoxic groups for 24 h and 48 h.The normal oxygen group was cultured at normal oxygen concentration environment while the low oxygen groups were cultured at 0.3%O2 concentration for 24 h and 48 h,respectively.qPCR and Western blot techniques were employed to detect the expression levels of GCRG213,Occludin,and Claudin-1 in mouse colon tissues and NCM-460 cells.Results The IHC results consistently showed that GCRG213 was expressed in the cytoplasm of intestinal epithelial cells but not in goblet cells in normal human small intestine,colon,and mouse colon tissues.In animal experiments,compared to the control group,the mRNA and protein expression levels of GCRG213 in mouse colon tissues were significantly upregulated in the experimental group(P<0.05 and P<0.01 respectively).Meanwhile,Occludin exhibited a significant downregulation at the protein level(P<0.05).In cell experiments,NCM-460 cells showed an upregulation of GCRG213 at both mRNA(P<0.001)and protein(P<0.01)levels after exposure to hypoxia,and

关 键 词：低压低氧 肠黏膜 紧密连接蛋白 GCRG213 LINE-1 

分 类 号：R776[医药卫生—眼科]