中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2014年
10期
747-752
,共6页
陆小霞%许敬%胡维琨%王莹%徐佳莉%陈和斌%陈鹏%董宗祈
陸小霞%許敬%鬍維琨%王瑩%徐佳莉%陳和斌%陳鵬%董宗祈
륙소하%허경%호유곤%왕형%서가리%진화빈%진붕%동종기
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
呼吸道閤胞病毒%半乳糖凝集素-9%Th17細胞%重組9型腺相關病毒
호흡도합포병독%반유당응집소-9%Th17세포%중조9형선상관병독
Respiratory syncytial virus%Galectin-9%Th17 cell%Recombinant adeno-associated virus serotype 9
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。
目的：觀察重組9型腺相關病毒（rAAV9）攜帶Galectin-9基因轉染小鼠外週血單箇覈細胞（PBMCs）的可行性，探討Galectin-9對呼吸道閤胞病毒（RSV）感染後輔助性T細胞（Th17）分化的影響。方法建立RSV感染小鼠模型，HE及PAS染色觀察RSV感染小鼠肺炎癥反應。分離小鼠PBMCs，利用攜帶Galectin-9基因的 rAAV9載體轉染 PBMCs，熒光顯微鏡觀察轉染效率；real-time PCR 檢測RORγt mRNA的錶達；流式細胞分析技術（ FACS）檢測Th17和Treg的比例；ELISA檢測細胞培養上清液中IL-10、IL-17及TGF-β1蛋白水平。結果與正常對照組相比，RSV感染組RORγt mRNA、Th17及IL-17的錶達均明顯升高（ P 均＜0．05）， Treg 及 IL-10的錶達均明顯降低（ P 均＜0．05）；使用 rAAV9-Galectin-9榦預PBMCs後，RSV＋Galectin-9組中RORγt mRNA、Th17及IL-17的錶達較RSV 組均顯著下降（P均＜0．05），Treg及IL-10的錶達均顯著升高（P均＜0．05）。結論 rAAV9-Galectin-9成功轉染PBMCs，可以通過調控Galectin-9通路影響RSV感染Th17免疫應答。
목적：관찰중조9형선상관병독（rAAV9）휴대Galectin-9기인전염소서외주혈단개핵세포（PBMCs）적가행성，탐토Galectin-9대호흡도합포병독（RSV）감염후보조성T세포（Th17）분화적영향。방법건립RSV감염소서모형，HE급PAS염색관찰RSV감염소서폐염증반응。분리소서PBMCs，이용휴대Galectin-9기인적 rAAV9재체전염 PBMCs，형광현미경관찰전염효솔；real-time PCR 검측RORγt mRNA적표체；류식세포분석기술（ FACS）검측Th17화Treg적비례；ELISA검측세포배양상청액중IL-10、IL-17급TGF-β1단백수평。결과여정상대조조상비，RSV감염조RORγt mRNA、Th17급IL-17적표체균명현승고（ P 균＜0．05）， Treg 급 IL-10적표체균명현강저（ P 균＜0．05）；사용 rAAV9-Galectin-9간예PBMCs후，RSV＋Galectin-9조중RORγt mRNA、Th17급IL-17적표체교RSV 조균현저하강（P균＜0．05），Treg급IL-10적표체균현저승고（P균＜0．05）。결론 rAAV9-Galectin-9성공전염PBMCs，가이통과조공Galectin-9통로영향RSV감염Th17면역응답。
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.
