表达EB病毒潜伏膜蛋白2的C3-GFP-LMP2肿瘤细胞模型构建及免疫效果初探  

A preliminary study on the immunological effect of C3-GFP-LMP2 model tumor cells expressing EBV LMP2

作　　者：孙立莹 王湛[1] 曾毅[1] SUN Li-ying;WANG Zhan;ZENG Yi(National Institute for Viral Disease Control and Prevention,Center for Disease Control and Prevention of China,State Key Laboratory for Infectious Disease Prevention and Control,Beijing 100052,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,传染病预防控制国家重点实验室,北京100052

出　　处：《中国病毒病杂志》2018年第3期209-215,共7页Chinese Journal of Viral Diseases

基　　金：国家重大创新药物研究开发项目(2013ZX09102114);国家高技术研究发展计划(863计划)(2012AA02A404)

摘　　要：目的构建表达EB病毒(Epstein-Barr virus,EBV)潜伏膜蛋白2(LMP2)与绿色荧光蛋白(GFP)的C3-GFP-LMP2肿瘤细胞模型,观察肿瘤形成及LMP2特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)杀伤肿瘤细胞的效果。方法利用Lenti-X HTX慢病毒包装系统,构建携带LMP2及GFP基因的C3-GFP-LMP2肿瘤细胞。用Western blot及流式细胞术鉴定EBV LMP2蛋白表达及细胞纯度。用iCELLigence系统实时监测LMP2特异性小鼠脾脏淋巴细胞杀伤肿瘤细胞效果。4×107个肿瘤细胞皮下接种C57BL/6小鼠,免疫组化及荧光显微镜观察肿瘤组织中EBV LMP2及GFP蛋白表达。结果 C3-GFP-LMP2肿瘤细胞可以很好地表达LMP2和GFP蛋白,连续传代30次,细胞纯度仍可达94.8%;且LMP2特异性小鼠脾脏淋巴细胞杀伤C3-GFP-LMP2后细胞指数明显下降;此外,接种肿瘤细胞的小鼠均可形成肿瘤,很好地表达LMP2和GFP蛋白。结论 C3-GFP-LMP2肿瘤细胞模型可以稳定地表达EBV LMP2和GFP蛋白,有效地被LMP2特异性CTL杀伤,接种小鼠可以很好地形成肿瘤组织。该研究为EBV LMP2相关疫苗的抗肿瘤效果评价提供了候选肿瘤细胞。Objective C3-GFP-LMP2 tumor cells were constructed as target cells to evaluate the tumor formation and the killing effects of specific CTL induced by LMP2-assosiated vaccines. Methods The lentiviral packaging system of Lenti-X HTX was used to construct C3-GFP-LMP2 tumor cells expressing LMP2 and GFP genes.The protein expression and cell purity of EBV LMP2 were determined by Western blot and flow cytometry.The LMP2-specific killing effects was detected using iCELLigence system.4×10^7 tumor cells were subcutaneously inoculated in C57 BL/6 mice,and the expressions of EBV LMP2 and GFP in tumor tissues were observed by immunohistochemistry and fluorescence microscope. Results C3-GFP-LMP2 tumor cells effectively expressed LMP2 and GFP protein;the purity of 30-passage tumor cells was as high as 94.8%,and the cell index significantly decreased in LMP2-specific mouse splenic lymphocytes mixed with C3-GFP-LMP2 target cells.In addition,the mice inoculated with C3-GFP-LMP2 cells formed tumors that effectively expressed LMP2 and GFP proteins. Conclusions C3-GFP-LMP2 cells stably expressing LMP2 and GFP produced tumors in mice,and could be effectively killed by LMP2-specific cytotoxic T lymphocyte（CTL）,suggesting that C3-GFPLMP2 was the putative target to assess the immune and anti-tumor effects of LMP2-related vaccines.

关 键 词：EB病毒潜伏膜蛋白2(LMP2) C3-GFP-LMP2 杀伤效果 成瘤 鼻咽癌 细胞毒性T淋巴细胞 

分 类 号：R392.12[医药卫生—免疫学]