Galectin-9基因转导干预呼吸道合胞病毒感染小鼠外周血 Th17细胞的研究  

Transfection of rAAV9-Galectin-9 into PBMCs inh ibits Th17 immune responses in mice with RSV infec- tion

作　　者：陆小霞[1] 许敬[1] 胡维琨[2] 王莹[1] 徐佳莉[1] 陈和斌[1] 陈鹏[1] 董宗祈[1] 

机构地区：[1]武汉市儿童医院呼吸内科,430016 [2]华中科技大学同济医学院眼科

出　　处：《中华微生物学和免疫学杂志》2014年第10期747-752,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金青年科学基金项目（81200006）;吴阶平医学基金（320.6750.1285）;湖北省自然科学基金（2010CDB08804）;武汉市卫生局科研项目（WX11B10）

摘　　要：目的：观察重组9型腺相关病毒（rAAV9）携带Galectin-9基因转染小鼠外周血单个核细胞（PBMCs）的可行性，探讨Galectin-9对呼吸道合胞病毒（RSV）感染后辅助性T细胞（Th17）分化的影响。方法建立RSV感染小鼠模型，HE及PAS染色观察RSV感染小鼠肺炎症反应。分离小鼠PBMCs，利用携带Galectin-9基因的 rAAV9载体转染 PBMCs，荧光显微镜观察转染效率；real-time PCR 检测RORγt mRNA的表达；流式细胞分析技术（ FACS）检测Th17和Treg的比例；ELISA检测细胞培养上清液中IL-10、IL-17及TGF-β1蛋白水平。结果与正常对照组相比，RSV感染组RORγt mRNA、Th17及IL-17的表达均明显升高（ P 均＜0．05）， Treg 及 IL-10的表达均明显降低（ P 均＜0．05）；使用 rAAV9-Galectin-9干预PBMCs后，RSV＋Galectin-9组中RORγt mRNA、Th17及IL-17的表达较RSV 组均显著下降（P均＜0．05），Treg及IL-10的表达均显著升高（P均＜0．05）。结论 rAAV9-Galectin-9成功转染PBMCs，可以通过调控Galectin-9通路影响RSV感染Th17免疫应答。Objective To evaluate the transfection efficiency of a recombinant adeno -associated virus serotype9 （ rAAV9 ） vector carrying Galectin-9 gene （ rAAV9-Galectin-9 ） into peripheral blood mononuclear cells （ PBMCs） and to analyze the effects of Galectin-9 on Th17 cell differentiation in a mouse model of respira -tory syncytial virus （RSV） infection.Meth ods A mouse model of RSV infection was established.Samples of lung sections were stained with HE or PAS to observe inflammatory infiltration.PBMCs were isolated from mice and transfected with rAAV9-Galectin-9.The transfection efficiency was evaluated by using fluorescence micro-scope.The expression of RORγt at mRNA level was detected by quantitative PCR.Flow cytometry analysis was performed to measure the levels of Th17 and Treg cells.ELISA was performed to measure the levels of IL-10, IL-17 and TGF-β1 in the supernatants of cell culture.Results Compared with control group, the mice with RSV infection showed an increased expression of ROR γt at mRNA level and elevated levels of T1h7 cells and IL-17 （all P〈0.05）, but decreased levels of Treg cells and IL-10 （all P〈0.05）.No significant difference with TGF-β1 level was observed.The expression of RORγt at mRNA level and the levels of Th17 cells and IL-17 were decreased in mice with RSV infection upon the transfection of Galectin -9 gene（all P 〈0.05）.Moreover, the levels of Treg cells and IL-10 were increased in mice from RSV＋Galectin-9 treated group （all P〈0.05）.No significant differences were observed between RSV infection group and RSV＋ZsGreen treated group.Conclusion rAAV9-Galectin-9 was successful transfected into PBMCs.rAAV9-Galectin9-could regulate Th17 immune re-sponses through Galectin-9 pathway during RSV infection.

关 键 词：呼吸道合胞病毒 半乳糖凝集素-9 TH17细胞 重组9型腺相关病毒 

分 类 号：R392[医药卫生—免疫学]