甘露糖结合凝集素对肠上皮细胞屏障功能的影响  

Role of mannose binding lectin in regulation of intestinal epithelial barrier in vivo and in vitro

作　　者：许超[1] 彭科[1] 王文生[1] 于敏[1] 孙力华[1] 杨桦[1] 

机构地区：[1]第三军医大学新桥医院普通外科,重庆400037

出　　处：《第三军医大学学报》2015年第11期1091-1095,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金重点项目(81330013);国家自然科学基金面上项目(81270451);国家自然科学基金青年科学基金(81200288);国家教育部创新团队项目基金(教技函[2013]59号)~~

摘　　要：目的观察甘露糖结合凝集素(mannose binding lectin,MBL)对人结肠癌细胞株Caco-2屏障功能的影响并探讨其分子机制。方法体内实验:取14只C57小鼠,按随机数字表法分为假手术组和LPS组(经腹腔注射LPS,构建肠炎症损伤模型),通过免疫组化和Real-time PCR观察MBL的变化情况,HE染色观察肠黏膜的损伤情况。体外实验:Caco-2细胞分为对照组、空转染组和MBL shRNA组(应用MBL干扰质粒转染Caco-2细胞),采用Real-time PCR、Western blot检测紧密连接蛋白的表达情况,并检测其跨上皮电阻(TER)。结果体内实验TER显示LPS组肠黏膜通透性(80.32±5.43)比假手术组(140.45±4.78)明显增加(P<0.01),HE染色显示LPS处理后肠黏膜完整性比假手术组降低,免疫组化和Real-time PCR检测结果显示LPS处理组MBL表达升高(P<0.05)。在体外实验中,我们成功构建MBL shRNA质粒模型,MBL shRNA组细胞中MBL的蛋白和mRNA表达[(0.34±0.09),(0.42±0.12)]较对照组及空转染组明显降低(P<0.05)。MBL shRNA组的紧密连接蛋白Occludin和Claudin-1的蛋白[(0.43±0.10)、(0.53±0.11)]和mRNA水平[(0.43±0.08)、(0.45±0.10)]明显低于其余组(P<0.05);MBL shRNA组TER值(80.33±7.64)降低较其余组明显(P<0.05)。结论下调MBL基因可减少肠上皮细胞紧密连接蛋白Occludin和Claudin-1 mRNA及蛋白水平的表达,减弱肠上皮细胞的屏障保护功能。harrier funetion of Objective To determine the effect of mannose binding lectin （MBL） on the epithelial Caco-2 cells and investigate the underlying mechanism. Methods In the in vivo study, 14 C57B1/6 mice were randomly divided into 2 groups, sham-operation and LPS groups. The LPS mice received intra-peritoneal injection of LPS to induce acute barrier injury. The expression of MBL at protein and mRNA levels in the intestinal mucosa was detected by immuno-histochemical assay and PCR, respectively. The morphological change of intestinal mucosa was observed by hematoxylin-eosin （HE） staining. In the in vitro study, Caco-2 cells were transfected with MBL-shRNA plasmid. Then, the expression of tight junction protein at protein and mRNA levels was measured by Western blotting and real-time PCR, respectively, and transepithelial electrical resistance （TER） was determined. Results In vivo study found that LPS group increased intestinal permeability compared with sham group （ TER value ： 80.32 ± 5.43 vs 140.45 ± 4.78, P 〈0.01 ）. HE staining showed LPS group had enhanced disruption of intestinal morphological structure than the sham group. Immuno-histochemical assay and real-time PCR assay indicated that the expression levels of MBL were apparently increased after LPS exposure （ P 〈 0.05 ）. After Caco-2 cells were transfected with MBL- shRNA plasmid, the expression of MBL at protein and mRNA levels was significantly decreased in MBL-shRNA cells （0.34 ± 0.09, 0.42 ± 0.12） than the control-shRNA ceils and the blank ceils （P 〈 0.05 ）. The protein and mRNA expression levels of Occludin （0.43 ±0.10, 0.43 ±0.08） and Claudin-1 （0.53 ±0.11, 0.45 ± 0.10） were obviously lower in MBL-shRNA cells than in control-shRNA and blank cells （P 〈 0.05 ）. The TER was 80.33 ± 7.64 in the MBL-shRNA cells, significantly lower than the other cells （P 〈 0.05 ）. Conclusion Down-regulation of MBL decreases the expression of tight junctions proteins Occludin and Claudin-1 at mRNA and prot

关 键 词：甘露糖结合凝集素 紧密连接蛋白 LPS 

分 类 号：R-322[医药卫生] R574.6