EB病毒潜伏膜蛋白LMP2A对人鼻咽癌细胞增殖与迁移特性的影响

目的:制备重组慢病毒载体pLMP2A,并检测其在人鼻咽癌细胞CNE1的表达情况及潜伏膜蛋白2A(latent membrane protein 2A,LMP2A)对人鼻咽癌细胞CNE1增殖?迁移?侵袭的影响?方法:利用DNA重组技术将EB病毒编码的LMP2A基因克隆到慢病毒表达载体plv-GFP中,通过酶切?测序验证,将重组慢病毒载体pLMP2A与包装质粒pdelta-8.91?pVSVG共转染人胚肾上皮细胞株293T,包装重组慢病毒LV-LMP2A,并感染人鼻咽癌细胞CNE1,经单克隆筛选后,用RT-PCR?免疫荧光和Western blot检测LMP2A在人鼻咽癌细胞CNE1的表达,CCK8法检测LMP2A对人鼻咽癌细胞CNE1增殖的影响,划痕实验?Transwell侵袭实验检测LMP2A对人鼻咽癌细胞CNE1迁移和侵袭的影响?结果:酶切及测序结果表明,成功制备了重组慢病毒载体pLMP2A;RT-PCR?Western blot?免疫荧光结果显示筛选出的细胞克隆能高表达EBV-LMP2A,CCK8结果显示LMP2A能促进人鼻咽癌细胞CNE1增殖,划痕实验结果显示LMP2A能促进人鼻咽癌细胞CNE1迁移,Transwell侵袭实验结果显示LMP2A能提高人鼻咽癌细胞CNE1侵袭能力?结论:成功制备了慢病毒载体pLMP2A,包装的慢病毒能够成功感染人鼻咽癌细胞系CNE1,使LMP2A基因得以高表达,并发现LMP2A能够促进人鼻咽癌细胞株CNE1增殖?迁移?侵袭,为进一步探讨EB病毒在鼻咽癌中的发病机制及基因治疗奠定了基础?

Effects of EB virus latent membrane protein 2A on human nasopharyngeal carcinoma cell proliferation and migration

Objective:To construct the recombinant lentiviral vector containing LMP2A gene and measure the expression level of LMP2A in human carcinoma cell line of CNE1, as well as the effects of LMP2A on proliferation, migration and invasion on human carcinoma cell line of CNE1. Methods: The EB virus LMP2A fragment was amplified by PCR and subcloned into the lentiviral vetor plv by recombinant DNA technology. The resultant lentivirus was confirmed by PCR, restriction enzyme digestion and DNA sequencing. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pLMP2A and packaging plasmid pdelta-8.91 and pVSVG. CNE1 cells were infected by plv-LMP2A, and the expression of LMP2A in CNE1 cells was confirmed by RT-PCR, immunofluorescence and Western blot by screening of monoclonal. CCK8 assay, Transwell invasion assay and Wound-healing assay were performed to determine the effects of LMP2A expression on the proliferation, invasion and migration. Results: The recombinant lentiviral vector carried the LMP2A gene was successfully constructed. Results of RT-PCR, immunofluorescence and western blot indicated that CNE1 transgenetic cells could high express EBV-LMP2A. The LMP2A gene-transfected carcinoma cells grew vigorously and rapidly and up-regulated the ability of migration and invasion. Conclusion: The recombinant lentiviral vector pLMP2A was successfully constructed, and could be used to transfect human carcinoma cell line of CNE1. LMP2A could be highly expressed and can significantly promote CNE1 proliferation, migration and invasion, which laid a foundation of study on the biological function of Epstein-Barr virus and its potential role in gene therapy of tumors. Key words] Epstein-Barr virus; latent membrane protein 2A; nasopharyngeal carcinoma; lentiviral vector