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

发布时间：2019-02-26 19:20

【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。
[Abstract]:Aim to construct eukaryotic expression vector p EGFP-N1-EMP1, of epithelial membrane protein 1 (EMP1) gene to investigate the effect of EMP1 gene on migration and invasion of human tongue squamous cell carcinoma cells. Methods the EMP1 gene was amplified by reverse transcription polymerase chain reaction (RT-(PCR) and ligated to the large fragment of the eukaryotic expression vector p-EGFP-N1. The recombinant plasmid p-EGFP-N1-EMP1 was constructed. After sequencing and identification, the recombinant plasmid p-EGFP-N1-EMP1 and the empty vector p-EGFP-N1 were transfected into human tongue squamous cell carcinoma cell line Tb3.1 by lipofectamine-mediated method. The expression of green fluorescent protein was observed under fluorescence microscope. Real-time fluorescence quantitative PCR assay was used to detect the over-expression of EMP1 at 24,48,72 hours after transfection. The effects of EMP1 overexpression on the migration and invasion ability of Tb3.1 cells were analyzed by Transwell migration and invasion assay. Results the full-length EMP1 gene was cloned successfully, and the green fluorescence expression of EMP1 gene was confirmed by sequencing analysis after the EMP1 gene was inserted into eukaryotic expression vector p-EGFP-N1,. The results of real-time fluorescence quantitative PCR showed that the expression of EMP1 in Tb3.1 cells was significantly higher than that in p-EGFP-N1 empty vector group after 24 h transfection with p-EGFP-N1-EMP1 recombinant plasmid. Transwell migration and invasion test showed that EMP1 overexpression inhibited the migration and invasion ability of Tb3.1 cells. Conclusion Eukaryotic expression vector of EMP1 has been successfully constructed, and the overexpression of EMP1 can inhibit the migration and invasion of tongue squamous cell carcinoma cells in vitro. It lays a foundation for further study on the role of EMP1 gene in the metastasis of oral squamous cell carcinoma (OSCC) and its molecular mechanism.
【作者单位】：天津市口腔医院·南开大学口腔医院中心实验室;天津市口腔医院·南开大学口腔医院颌面外科;
【基金】：天津市卫计委基金(2013KZ053)~~
【分类号】：R739.86

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