上皮膜蛋白1真核表达载体的构建及其对人舌鳞状细胞癌细胞迁移和侵袭的影响  

Construction of epithelial membrane protein 1 eukaryotic expression vector and its influence on migration and invasion of human oral tongue squamous carcinoma cells

作　　者：代晓华[1] 张军[2] 邹慧儒[1] 连小丽[1] 李燕妮[1] 王冠华[1] 颜艳[1] 

机构地区：[1]天津市口腔医院.南开大学口腔医院中心实验室,天津300041 [2]天津市口腔医院.南开大学口腔医院颌面外科,天津300041

出　　处：《华西口腔医学杂志》2016年第4期398-403,共6页West China Journal of Stomatology

基　　金：天津市卫计委基金(2013KZ053)~~

摘　　要：目的构建上皮膜蛋白1(EMP1)基因真核表达载体p EGFP-N1-EMP1,探讨EMP1基因对人舌鳞状细胞癌细胞迁移和侵袭的影响。方法采用逆转录聚合酶链式反应(PCR)扩增EMP1基因,双酶切法连接至真核表达载体p EGFP-N1双酶切产物大片段,构建p EGFP-N1-EMP1重组质粒。经测序鉴定后,通过脂质体介导法将p EGFP-N1-EMP1重组质粒和p EGFP-N1空载体转染至人舌鳞状细胞癌Tb3.1细胞,荧光显微镜下观察转染后绿色荧光蛋白的表达情况,实时荧光定量PCR法检测转染后24、48、72 h的EMP1过表达水平。利用Transwell迁移及侵袭实验分析EMP1过表达对Tb3.1细胞迁移及侵袭能力的影响。结果成功克隆EMP1全长基因,经测序分析证实将EMP1基因准确插入真核表达载体p EGFP-N1,转染后细胞可见绿色荧光表达。实时荧光定量PCR结果显示,p EGFP-N1-EMP1重组质粒转染24 h组,Tb3.1细胞中EMP1表达量显著高于p EGFP-N1空载体组、野生型细胞组以及重组质粒转染48 h和72 h组。Transwell迁移及侵袭实验结果显示,EMP1过表达抑制了Tb3.1细胞的迁移和侵袭能力。结论成功构建了EMP1真核表达载体,并在体外证实了EMP1过表达可抑制舌鳞状细胞癌细胞的迁移和侵袭能力,为进一步研究EMP1基因在口腔鳞状细胞癌转移中的作用及其分子机制奠定了基础。ObjectiveThis study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 （EMP1） and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells.Methods The human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72 h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay.Results The full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indi-cated that the expression of EMP1 at 24 h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells.Conclusion The eukaryotic expres-sion vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cellsin vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism.

关 键 词：上皮膜蛋白1 舌鳞状细胞癌细胞 载体 转染 细胞迁移 细胞侵袭 

分 类 号：R739.86[医药卫生—肿瘤]