闭合蛋白的胞外环缺失对小鼠TM4细胞紧密连接的影响分析  

Deletion of extracellular loops of occludin affects the tight junction of TM4 cells in mice

作　　者：谭小方 陈德宇[3] 丁家怡 吴洁[1] TAN Xiao-fang;CHEN De-yu;DING Jia-yi;WU Jie(State Key Laboratory of Reproductive Medicine,Maternity and Child Health Care Hospital of Jiangsu Province,The First Affiliated Hospital of Nanjing Medical University/Jiangsu Provincial People's Hospital,Nanjing,Jiangsu 210029,China;Center of Reproductive Medicine,Maternity and Child Health Care Hospital of Nantong Universi-ty,Nantong,Jiangsu 226000,China;School of Biological and Food Engineering,Fuyang Normal University,Fuyang,Anhui 236037,China)

机构地区：[1]南京医科大学第一附属医院/江苏省人民医院/江苏省妇幼保健院生殖医学国家重点实验室,江苏南京210029 [2]南通大学附属妇幼保健院生殖医学中心,江苏南通226000 [3]阜阳师范大学食品与生物工程学院,安徽阜阳236037

出　　处：《中华男科学杂志》2020年第8期675-680,共6页National Journal of Andrology

基　　金：国家自然科学基金(81771567);安徽省高校自然科学重大、重点研究项目(KJ2017A338);南通市科技计划项目(GLZ16078)。

摘　　要：目的:分析闭合蛋白的2个胞外环结构域在小鼠TM4细胞紧密连接中的功能。方法:利用基因工程的方法,分别或者同时缺失掉闭合蛋白的2个胞外环,将缺失了胞外环3个闭合蛋白基因序列,分别克隆入pcDNA3.1表达载体,再转入TM4细胞。用RT-PCR和Western印迹分析闭合蛋白表达量,用紧密连接的体外细胞模型来分析闭合蛋白的胞外环对紧密连接程度的影响。结果:测序结果表明pcDNA3.1-occludinΔOCC1、pcDNA3.1-occludinΔOCC2和pcDNA3.1-occludinΔOCC1+OCC2表达载体已经构建成功。RT-PCR和Western印迹结果显示3个缺失组闭合蛋白的mRNA和蛋白质的表达量都显著高于对照组。体外紧密连接模型分析结果表明闭合蛋白2个胞外环都能够增加TM4的紧密连接程度,其中,pcDNA3.1-occludinΔOCC1转染组每天的大分子通过率均比pcDNA3.1-occludinΔOCC2转染组显著降低(P<0.05),表明闭合蛋白第二个胞外环对紧密连接的影响程度比第一个胞外环要高。结论:闭合蛋白2个胞外环都能够影响小鼠TM4细胞紧密连接的程度,并且第二个胞外环对紧密连接的影响程度比第一个胞外环要高。Objective: To analyze the functions of the two extracellular loops of occludin in the tight junction of TM4 cells in mice. Methods: Using genetic engineering, we separately or simultaneously deleted two extracellular loops of occludin, cloned the three occludin genes without extracellular loops into the pcDNA3.1 expression vector, and transfected them into TM4 cells. Then we determined the expression of occludin by RT-PCR and Western blot, and analyze the effects of the extracellular loops of occludin on the tight junction of the TM4 cells with the in vitro cell line model. Results: The results of sequencing showed that the expression vector of pcDNA3.1-occludin Δ OCC1, pcDNA3.1-occludin Δ OCC2 and pcDNA3.1-occludin Δ OCC1 + OCC2 was constructed successfully. The mRNA and protein expressions of occludin in the non-extracellular loop groups were significantly higher than in the control group. Both the extracellular loops of occludin increased the tight junction of the TM4 cells. The macromolecular permeability in the TM4 cells was significantly lower in the pcDNA3.1-occludin Δ OCC1 than in the pcDNA3.1-occludin Δ OCC2 group(P < 0.05), indicating a higher impact of the second than the first extracellular loop on the tight junction of the TM4 cells. Conclusion: Both of the two extracellular loops of occludin can affect the tight junction of TM4 cells, the second even more significantly than the first one.

关 键 词：血-睾屏障 闭合蛋白 胞外环结构域 紧密连接 TM4细胞 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]