發(fā)布時(shí)間：2019-02-26 19:20

【摘要】：目的構(gòu)建上皮膜蛋白1(EMP1)基因真核表達(dá)載體p EGFP-N1-EMP1,探討EMP1基因?qū)θ松圜[狀細(xì)胞癌細(xì)胞遷移和侵襲的影響。方法采用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增EMP1基因,雙酶切法連接至真核表達(dá)載體p EGFP-N1雙酶切產(chǎn)物大片段,構(gòu)建p EGFP-N1-EMP1重組質(zhì)粒。經(jīng)測序鑒定后,通過脂質(zhì)體介導(dǎo)法將p EGFP-N1-EMP1重組質(zhì)粒和p EGFP-N1空載體轉(zhuǎn)染至人舌鱗狀細(xì)胞癌Tb3.1細(xì)胞,熒光顯微鏡下觀察轉(zhuǎn)染后綠色熒光蛋白的表達(dá)情況,實(shí)時(shí)熒光定量PCR法檢測轉(zhuǎn)染后24、48、72 h的EMP1過表達(dá)水平。利用Transwell遷移及侵襲實(shí)驗(yàn)分析EMP1過表達(dá)對Tb3.1細(xì)胞遷移及侵襲能力的影響。結(jié)果成功克隆EMP1全長基因,經(jīng)測序分析證實(shí)將EMP1基因準(zhǔn)確插入真核表達(dá)載體p EGFP-N1,轉(zhuǎn)染后細(xì)胞可見綠色熒光表達(dá)。實(shí)時(shí)熒光定量PCR結(jié)果顯示,p EGFP-N1-EMP1重組質(zhì)粒轉(zhuǎn)染24 h組,Tb3.1細(xì)胞中EMP1表達(dá)量顯著高于p EGFP-N1空載體組、野生型細(xì)胞組以及重組質(zhì)粒轉(zhuǎn)染48 h和72 h組。Transwell遷移及侵襲實(shí)驗(yàn)結(jié)果顯示,EMP1過表達(dá)抑制了Tb3.1細(xì)胞的遷移和侵襲能力。結(jié)論成功構(gòu)建了EMP1真核表達(dá)載體,并在體外證實(shí)了EMP1過表達(dá)可抑制舌鱗狀細(xì)胞癌細(xì)胞的遷移和侵襲能力,為進(jìn)一步研究EMP1基因在口腔鱗狀細(xì)胞癌轉(zhuǎn)移中的作用及其分子機(jī)制奠定了基礎(chǔ)。
[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.
【作者單位】： 天津市口腔醫(yī)院·南開大學(xué)口腔醫(yī)院中心實(shí)驗(yàn)室;天津市口腔醫(yī)院·南開大學(xué)口腔醫(yī)院頜面外科;
【基金】：天津市衛(wèi)計(jì)委基金(2013KZ053)~~
【分類號】：R739.86

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