微小RNA-21、紧密连接蛋白occludin 和ROCK1与幼鼠肠屏障功能障碍的关系  

The Relationship Between MiRNA-21 and Tight Junction Protein Occludin ROCK1 and Intestinal Barrier Dysfunction in Young Rats

作　　者：郭小刚 罗培燕 GUO Xiao-gang;LUO Pei-yan(Chongqing Qianjiang Central Hospital,Chongqing 409000,China)

机构地区：[1]重庆市黔江中心医院

出　　处：《标记免疫分析与临床》2019年第11期1940-1944,1971,共6页Labeled Immunoassays and Clinical Medicine

摘　　要：目的研究微小RNA-21、紧密连接蛋白occludin和ROCK1与幼鼠肠屏障功能障碍的关系。方法将36只4周龄小鼠随机分为2组:假手术组、肠屏障功能障碍组。通过苏木精和伊红染色观察肠系膜病理学变化;通过Chiu病理评分评估肠系膜切片的组织学改变程度;通过双荧光素酶报告系统测量ROCK1荧光素酶活性;通过RT-qPCR和蛋白质免疫印迹检测miRNA-21、ROCK1和occludin mRNA和蛋白表达量;通过ELISA试剂盒检测炎症因子(TNF-α、IL-6)、iFABP和MPO水平。结果在假手术组中,在肠黏膜中仅观察到少量淋巴细胞浸润,肠黏膜正常,厚度均匀,绒毛排列整齐,上皮完整。在肠屏障功能障碍组中,观察到肠绒毛上皮和椎板分离,肠绒毛破裂,出血和溃疡,固有层中有炎性细胞,黏膜层坏死。与假手术组相比,肠屏障功能障碍组Chiu病理评分升高(P<0.05);miRNA-21与ROCK1的3′UTR直接相互作用,ROCK1突变体组(0.98±0.20)3′UTR转染的细胞中ROCK1的荧光活性与ROCK1野生型组相比升高(P<0.05);与假手术组相比,肠屏障功能障碍组miRNA-21 mRNA表达量增加,而ROCK1和occludin mRNA和蛋白表达量均降低(P<0.05);肠屏障功能障碍组TNF-α、IL-6、iFABP和MPO水平高于假手术组(P<0.05)。结论缺血改变了模型中miRNA-21和ROCK1的表达,miRNA-21通过靶向ROCK1的3′UTR影响occludin表达,影响肠道细胞旁通透性。Objective To study the relationship between microRNA-21 and tight junction protein occludin ROCK1 and intestinal barrier dysfunction in young rats.Methods 36 4-week-old mice were randomly divided into two groups:the sham-operated group(young rats underwent midline laparotomy,superior mesenteric artery occlusion was not observed,n=18)and the intestinal barrier dysfunction group(young rats underwent midline laparotomy,clamped superior mesenteric artery by non-traumatic vascular clamp,ischemia induction and then perfusion for 6 hours,n=18).Hematoxylin and eosin staining were used to observe the pathological changes of mesentery.The degree of histological changes of mesenteric sections was evaluated by Chiu pathological score.ROCK1 luciferase activity was measured by dual luciferase reporter system.miRNA-21 and ROCK1 were detected by RT-qPCR and western blotting,as well as occludin mRNA and protein expression levels.Inflammatory factors(TNF-α,IL-6),iFABP and MPO levels were detected by ELISA kit.Results In the sham operation group,only a small amount of lymphocyte infiltration was observed in the lower intestinal mucosa.The intestinal mucosa was normal,with uniform thickness,neatly arranged villi and intact epithelium.In the bowel barrier dysfunction group,we observed villous epithelial and lamina separation,villous rupture,bleeding,ulceration,inflammatory cells in the lamina propria,and mucosal necrosis.Compared with the sham operation group(0.92±0.14),the pathological score of Chiu in the intestinal barrier dysfunction group was increased(P<0.05).miRNA-21 could directly interact with the 3’utr of ROCK1,and the fluorescence activity of ROCK1 in the 3’utr transfected cells of the ROCK1 mutant group(0.98±0.20)was increased compared with that of the wild-type group(0.47±0.25)(P<0.05).Compared with the mice in the sham operation group,the expression of miRNA-21 mRNA in the intestinal barrier dysfunction group increased,while the expressions of ROCK1,occludin mRNA and protein decreased(P<0.05).The levels of TNF-a

关 键 词：肠屏障功能障碍 紧密连接蛋白 微小RNA-21 缺血再灌注 荧光素酶活性 

分 类 号：R73[医药卫生—肿瘤]