流式细胞术检测分析人巨细胞病毒核酸疫苗诱导宿主细胞免疫效应  

Study on the detection host cellular immunity after inoculation of human cytomegalovirus DNA vaccine by flow cytometry

作　　者：方风琴[1] 张玥[1] 钱春艳[1] 卫蓓文[1] 史桂英[2] 季育华[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院检验科,上海200025 [2]上海交通大学医学院细胞生物学教研室,上海200023

出　　处：《检验医学》2007年第3期240-244,共5页Laboratory Medicine

基　　金：上海市教委科研重点资助项目(04BA04)

摘　　要：目的探讨人巨细胞病毒(HCMV)pp65核酸疫苗对宿主细胞免疫功能的诱导效应。方法用自行构建的含HCMVpp65基因的重组质粒免疫小鼠,用流式细胞仪检测免疫小鼠脾脏T淋巴细胞亚群的变化以及CD8+T细胞分泌干扰素γ(IFN-γ)和白细胞介素(IL)-4的情况,并以四甲基偶氮唑盐(MTT)增殖试验作补充验证。结果接种重组质粒组与仅接种空质粒或生理盐水对照组小鼠的免疫效应不同,表现为CD8+T细胞百分率增加(P<0.05),分泌细胞因子IFN-γ和IL-4的CD8+T细胞百分率亦增高(P<0.05),两者的平均荧光强度值增高更为显著(P<0.05)。免疫小鼠的脾脏T细胞经HCMVpp65蛋白刺激后,实验组的刺激指数(SI)较对照组明显增高(P<0.05)。结论HCMVpp65核酸疫苗接种可诱导宿主的细胞免疫反应,倾向于表现为细胞毒性T细胞(Tc)1和Tc2的混合应答以及脾脏T细胞的增殖效应。Objective To study the host cellular immunity response to DNA vaccine of human cytomegalovirus （HCMV） pp65 gene. Methods Plasmids （PVAX1-pp65） with HCMVpp65 gene, which were recombined, construc-ed,propagated and purified, were prepared and used for immunizing the BALB/C mice. After immunization,the spleen T lymphocyte suspension were collected. Then the change of T lymphocyte subunits and interferon-γ, （IFN-γ）/interleukin （IL）-4 -secreting state in CD8 ^＋ T lymphocyte were detected by flow cytometry, while the specific lymphoproliferation was observed by methyl thiazolylte trazolium （MTF） colorimetric assay. Results The mice immunized by PVAXl-pp65 shew different immunological reactions compared to the control mice injected with PVAX1 or normal saline. The percentage increased of CD8^＋ T cell and the percentage of CD8^＋ T cell secreting IFN-γ and IL-4 also increased （P 〈 0.05） and average fluorescence intensity were all increased （P 〈 0.05）. In addition, the stimulation index of experimental group had statistical significance comparing to control group （P 〈 0.05）. Conclusions DNA vaccine of HCMVpp65 could induce the cellular immunity of the host. It expressed by the mixed immunization of cytotoxic T cell （Tc） 1 and Tc2, in addition to proliferation effects of the spleen T lymphocytes.

关 键 词：PP65 人巨细胞病毒 DNA疫苗 特异性细胞免疫 细胞因子 流式细胞术 

分 类 号：R446.61[医药卫生—诊断学]