腺病毒介导MOMP基因转染对树突状细胞免疫功能的影响  

Functional effects of dendritic cells transfected with recombinant adenovirus carrying Chlamydia trachomatis serovar E MOMP gene

作　　者：吕慧[1,2] 王红[1] 李湘新[3] 齐眉[1] 刘娟[1] 郑燕[1] 李靖[1] 赵蕾[1] 杨熙[4] 于修平[1] 赵蔚明[1] 

机构地区：[1]山东大学医学院微生物学教研室,济南250012 [2]山东省疾病预防控制中心,济南250014 [3]山东大学齐鲁医院血液科 [4]加拿大曼尼托巴大学医学微生物系

出　　处：《中华微生物学和免疫学杂志》2008年第1期55-60,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金对外交流与合作项目（30310403165）;国家自然科学基金资助项目（30271193）

摘　　要：目的探讨腺病毒（Ad）介导E型沙眼衣原体（Ct）主要外膜蛋白（MOMP）基因转染对树突状细胞（DC）的表型及体内免疫功能的影响。方法用小鼠重组粒细胞-巨噬细胞集落刺激因子（GM-CSF）及白细胞介素4（IL-4）从小鼠骨髓干细胞中诱导培养DC，并用大肠杆菌脂多糖（LPS）促进DC的成熟，流式细胞仪检测DC表型。用不同感染滴度（MOI）的含增强绿色荧光蛋白（EGFP）基因的重组Ad转染DC，荧光显微镜下观察EGFP的表达细胞百分率，选择最佳MOI；用最佳MOI的含MOMP基因的重组腺病毒（Ad-MOMP）转染DC，流式细胞术检测转染前后DC表型变化，并用活细胞计数试剂盒8（CCK-8）检测转染前后DC刺激同种淋巴细胞增殖的能力；ELISA检测转染前后DC分泌细胞因子及DC、T细胞共同培养上清中细胞因子的水平。Ad-MOMP转染DC经尾静脉免疫小鼠，ELISA检测其脾脏淋巴细胞分泌的细胞因子。结果诱导的DC形态典型，表面高表达CD11c、MHCⅡ类分子，中度表达CD80分子。MOI=1000为重组Ad转染DC最佳滴度，此时90％以上的DC表达荧光，Ad-MOMP转染DC后能检测到MOMP的表达。Ad-MOMP转染对DC表面的特征性表型CD11c无影响，而CD80及MHCⅡ表达上调；转染后的DC分泌大量IL-12，具有较强的刺激同种异体淋巴细胞增殖的能力，并刺激T细胞分泌大量IFN-γ。Ad-MOMP转染DC后尾静脉免疫小鼠，其脾细胞产生高水平的IFN-γ。结论Ad载体能介导外源MOMP基因在DC中的表达，Ad-MOMP转染DC后MOMP基因的表达能增强DC抗原提呈功能，促进DC活化，转染后的DC与T细胞共孵育能诱导T细胞向TH1细胞分化，体内实验表明Ad-MOMP转染DC能诱导衣原体特异性TH1反应。这为Ad-MOMP转染的DC疫苗用于免疫治疗提供了理论依据和技术基础。Objective To study the effects of dendritic cells （DC） transfected with recombinant adenovirus （Ad） carrying Chlamydia trachomatis（Ct） serovar E major outer membrane protein （MOMP） gene. Methods Bone marrow-derived DC were generated from female BALB/c mice and induced with recombinant cytokines GM-CSF and IL-4. After stimulated by Escherichia coli lipopolysaccharide （LPS） , DC were infected with Ad encoding enhanced green fluorescent protein （EGFP） at various values of multiplicity of infection （MOI）. The percentage of DC expressing EGFP protein was analyzed by fluorescence microscope, and the optimal MOI was selected. According to the optimal MOI, DC were transfected with recombinant Ad carrying Ct serovar E MOMP gene and then were used for analysis of the phenotypic markers by flow cytometry and the stimulatory capacity in T-cell proliferation assay. Cytokine production on transfected DC and on T cells stimulated by transfected DC was measured by ELISA. Mice were adoptively immunized by intravenous （vena caudalis） injection and cytokines secreted by spleen cells of mice were measured by ELISA. Results Cultured cells displayed a typical DC appearance which expressed high levels of CD11 c and MHC Ⅱ surface markers, moderate levels of CD80. About 90% DC expressed EGFP after transfection at the MOI of 1000. DC infected with recombinant Ad expressed MOMP, up-regulated the expression of DC surface marker MHC Ⅱ and CD80, secreted elevated IL-12, obviously enhanced the capacity to stimulate the proliferation of allogenetic T cells, and stimulated T cells to secrete IFN-γ at much higher level. Spleen cells of mice immunized with transfected DC produced elevated concentrations of IFN-γ. Conclusion MOMP gene, which was carried by Ad vectors, could be transferred into DC. The expression of MOMP gene activated DC, enhanced antigen presentation of DC, and resulted in the polarization of TH1 cells. Transfected DC can induce Chlamydia specific TH1 response in mice.

关 键 词：沙眼衣原体 主要外膜蛋白基因 腺病毒载体 树突状细胞 

分 类 号：R686[医药卫生—骨科学]