文章摘要
饶月丽,沈 定,陈伟民,许冬亚,赵 薇,丁贵鹏,严 杰.Trop2过表达对人胃黏膜上皮细胞GES-1迁移能力的影响及机制[J].南京医科大学学报,2016,(11):1300~1305
Trop2过表达对人胃黏膜上皮细胞GES-1迁移能力的影响及机制
Effects of over-expression of Trop2 on migration of human gastric mucosal epithelial cells and its mechanism analyses
投稿时间:2016-09-05  
DOI:10.7655/NYDXBNS20161104
中文关键词: Trop2  GES-1  迁移  上皮-间质转化
英文关键词: Trop2  GES-1  migration  epithelial-mesenchymal transition
基金项目:
作者单位
饶月丽 浙江大学医学院,浙江 杭州 310058
解放军第一一七医院输血科,浙江 杭州 310013 
沈 定 解放军第一一七医院输血科,浙江 杭州 310013 
陈伟民 解放军第一一七医院输血科,浙江 杭州 310013 
许冬亚 解放军第一一七医院输血科,浙江 杭州 310013 
赵 薇 南京医科大学病理学系,江苏 南京 211166 
丁贵鹏 南京医科大学病理学系,江苏 南京 211166 
严 杰 浙江大学医学院病原微生物系,浙江 杭州 310058 
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中文摘要:
      目的:观察Trop2过表达对人胃黏膜上皮细胞GES-1迁移能力的影响,并对其作用机制进行初步探讨?方法:real-time PCR和Western blot检测GES-1细胞中Trop2 mRNA和蛋白的表达水平;构建过表达Trop2质粒及空载对照质粒并转染GES-1细胞,检测转染效率;细胞划痕实验和Transwell迁移实验检测过表达Trop2后GES-1细胞迁移能力的变化情况;Western blot检测过表达Trop2后GES-1细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子[上皮细胞标志E-钙黏附蛋白(E-cadherin),间皮细胞标志纤维连接蛋白(fibronectin)?波形蛋白(vimentin)]的变化情况?结果:相比胃癌细胞株BGC823和MGC803,Trop2在GES-1细胞中的mRNA和蛋白表达水平均较低;过表达Trop2的质粒转染GES-1后,Trop2的mRNA表达水平与未转染组相比提高(6.18 ± 0.52)倍,而空载对照组和未转染组相比无明显变化,蛋白水平的变化情况和mRNA相似;细胞划痕实验表明,转染72 h后,过表达组细胞迁移率达到95%,空载对照组细胞迁移率为50%,两组相比差异有统计学意义(P﹤0.05);Transwell迁移实验发现,转染24 h后,过表达组穿膜细胞数为(87 ± 6)个,而空载对照组为(41 ± 6)个,两组相比差异有统计学意义(P﹤0.05);Western blot检测过表达Trop2后,随着Trop2蛋白表达量增高,GES-1细胞E-cadherin表达下调,fibronectin和vimentin表达上调?结论:Trop2过表达可以提高胃黏膜上皮细胞的迁移能力,其机制与Trop2促进胃黏膜上皮细胞EMT有关?
英文摘要:
      Objective:To study the effecs of human trophoblast cell surface glycoprotein(Trop2) overexpression on migration of the human gastric mucosal epithelial cells and preliminary mechanism analyses. Methods:We explored mRNA and protein expression levels of Trop2 in GES-1 cell line by real-time RT-PCR and Western blot. OE-Trop2 and VE-Trop2 plasmids were constructed and then transfected into GES-1 cell line,and the efficiency of transfection was detected. Cell wound healing assay and Transwell migration assay were performed to detect the change of the migration in GES-1 after transfected OE-Trop2 plasmids. The change of surface markers(E-cadherin,fibronectin,and vimentin)of epithelial-mesenchymal transition(EMT)in GES-1 cell line were detected by Western blot after Trop2 overexpression. Results:Compared with gastric cancer cell lines BGC823 and MGC803,the mRNA and protein expression levels of Trop2 in GES-1 were lower. After OE/VE-Trop2 plasmid transfected in GES-1,the mRNA expression level of Trop2 in the OE-Trop2 group was 6.18 ± 0.52 times higher than that of the control group,whereas no differences were detected between the control group and the VE-Trop2 group. Meanwhile,the protein expression model of Trop2 was similar to that of the mRNA expression. After transfected plasmids 72 h,cell wound healing assay showed that the migration rate of GES-1 was 50% in the VE-Trop2 group and 95% in the OE-Trop2 group(P < 0.05). After transfected OE-Trop2 plasmids 24 h,transwell migration assay showed that the number of migration cell was 41 ± 6 in the VE-Trop2 group,whereas 87 ± 6 in the OE-Trop2 group(P < 0.05). Western-blot assay showed that the expression level of E-cadherin in GES-1 was down-regulated;meanwhile,fibronectin and vimentin were up-regulated after transfected OE-Trop2. Conclusion:Trop2 overexpression could promote migration of GES-1 cell line through inducing EMT phenomenon.
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