HPV18型E2蛋白高表达对巨噬细胞凋亡及其分泌功能的影响  

Effect of over expression of HPV18 E2 protein on apoptosis and secretion of macrophages

作　　者：彭俊[1] 朱翠明[1] 余敏君[1] 曹清香[1] 王鑫[1] 万艳平[1] 

机构地区：[1]南华大学病原生物学研究所,湖南衡阳421001

出　　处：《细胞与分子免疫学杂志》2008年第2期136-138,141,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：湖南省教育厅资助项目(02C391)

摘　　要：目的:研究HPV18E2及其N端(TAD)、C端(DBD)与GFP融合蛋白瞬时高表达对巨噬细胞(MΦ)凋亡及分泌活性的影响,为进一步研究E2蛋白在HPV18致癌机制中的作用奠定实验基础。方法:通过PCR从现有真核表达载体pEG-FP-C1/E2上分别扩增出TAD、DBD基因片段,并构建真核表达载体pEGFP-C1/TAD、pEGFP-C1/DBD。将3种真核表达载体及pEGFP-C1分别转染MΦ,用倒置荧光显微镜观察它们的表达与定位,并以抗GFP抗体为一抗作Westernblot检测它们的表达。通过3种融合蛋白在MФ内的瞬时高表达,在转染48h后分别检测各组细胞培养基中TNF-α和IL-1β的含量,并收集MΦ经染色以及流式细胞术(FCM)观察检测其凋亡。结果:GFP-E2融合蛋白主要表达于细胞核,细胞质内也有表达,而GFP-DBD融合蛋白仅表达于细胞核内,GFP-TAD仅表达于细胞质。GFP-E2、GFP-TAD融合蛋白在MΦ内高表达后MΦ凋亡率上升,细胞因子TNF-α和IL-1β分泌量增加,且GFP-TAD作用强于GFP-E2。而EGFP-DBD无此作用。结论:HPV18E2及其TAD与EGFP融合蛋白瞬时高表达可诱导MΦ凋亡并上调其分泌细胞因子TNF-α和IL-1β。AIM： To study the effect of the over expression of GFP-E2, GFP-TAD （ N-extremity domain of HPV18 E2） and GFP-DBD （C-extremity domain of HPV18 E2） on the apoptosis and secretion of macrophages and to further explore the contribution of E2 gene to the uterine cervix cancer. METHODS： TAD or DBD gene was amplified from pEGFP-C1/HPV18 E2 by PCR respectively and then cloned into pEGFP-C1 vector. After the transfection of recombinant plasmids or pEGFP-C1 into the macrophages, their expression was examined by fluorescent microscopy and Western blot. The cytokine content of TNF-α or IL-1β in the culture medium was tested quantitatively with ELISA kit respectively. The stained macrophages were observed and their apoptosis rate was tested by flow cytometry. RESULTS： After transfected into macrophages, GFP-E2 fusion protein was mainly located in cytoplasma while GFP-DBD fusion protein was completely located in nuclei and GFP-TAD fusion protein was completely located in cytoplasma. The overexpression of GFP-E2 or GFP-TAD increased the level of TNF-α and IL-1β and upregulate the apoptosis rate of macrophages. Furthermore, the effect of GFP-TAD was obvious except on IL-1β level but the overexpression of GFP-DBD did not show the same effect. CONCLUSION： The overexpression of GFP-E2 or GFP-TAD fusion protein can induce the apoptosis macrophages and upregulate TNF-α or IL-1β secretion of macrophages.

关 键 词：人乳头瘤病毒18型 E2蛋白 巨噬细胞 凋亡 细胞因子 

分 类 号：R373.9[医药卫生—病原生物学]