HPV18型E2蛋白高表达对巨噬细胞凋亡及其分泌功能影响.docVIP

HPV18型E2蛋白高表达对巨噬细胞凋亡及其分泌功能影响 　 作者：彭俊, 朱翠明, 余敏君, 曹清香, 王鑫, 万艳平 【摘要】 　　目的: 研究HPV18 E2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pEGFP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Western blot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48 h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: HPV18 E2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。 【关键词】 人乳头瘤病毒18型 E2蛋白 巨噬细胞 凋亡 细胞因子 　　[Abstract] 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 prot