IL-17R-FcγRⅡb基因体外诱导树突状细胞表达及其对淋巴细胞增殖活化的影响

Effect of IL-17R-FcγRⅡb gene on the expression of dendritic cells in vitro and on the activation of lymphocyte's proliferation

目的:构建编码人全长IL-17R与FcγRⅡb双基因表达重组腺病毒载体并对其特性进行鉴定,探讨阻断其信号调节通路对妊娠哮喘发病的影响。方法:将扩增出的拼接基因片段定向克隆于pAdTrack-CMV中经与pAdeasy-1同源重组,鉴定正确的阳性重组子通过脂质体介导转入AD293细胞,荧光显微镜观察绿色荧光(GFP)信号并采用PCR法对其进行鉴定;纯化腺病毒感染体外细胞因子协同诱导培养的妊娠哮喘模型小鼠骨髓来源树突状细胞(DC),利用RT-PCR、Western免疫印迹检测IL-17R和FcγRⅡb表达;流式细胞术、CCK-8、ELISA分析重组腺病毒的修饰对DC表面分子表达,刺激T细胞增殖抑制变化和MLR上清中IFN-γ、IL-4、IL-17分泌水平影响。结果:构建的重组腺病毒载体插入序列完全正确;经GFP表达和PCR检测证实成功包装获得高滴度重组腺病毒;感染DCs的GFP阳性细胞数达到85%;在mRNA及蛋白水平均可同时转录和表达出符合预期的IL-17R和FcγRⅡb。CD11c阳性表达率在各组间无显著差异;与对照细胞及EGFP-DCs相比,经IL-17RFcγRⅡb修饰的DC表面CD80表达增高,而CD86表达减少(P<0.05);IL-17R-FcγRⅡb-DCs相同浓度比例刺激T细胞增殖水平均明显受抑(P<0.05);转染载体共培养物上清液中Th2/Th17类细胞因子IL-4、IL-17水平下降,Th1类细胞因子IFN-γ升高(P<0.05),IFN-γ/IL-4/IL-17相对比值增加(P<0.05)。结论:IL-17R-FcγRⅡb融合基因体外阻断CD86/CD28共刺激通路可诱导DC免疫耐受状态,抑制抗原特异性T细胞增殖并影响Th1/Th2/Th17亚群分化,有望在妊娠哮喘免疫耐受缺陷和缓解气道炎症反应中发挥作用。

Objective： To construct the recombinant adenovirus vector which co-express human IL-17R and FcγRⅡb gene and further identify its characteristics. This study also explores the effect of this recombinant adenovirus vector on the morbidity of asthma during pregnancy,while blocking the signal regulation of IL-17R-FcγRⅡb. Methods： The splicing fragment of IL-17R and FcγRⅡb gene were amplified and sub-cloned to pAdTrack-CMV shuttle plasmid and then was homologously recombined with backbone plasmid pAdeasy-1. After digested and linearized,the recombinant was transfected into AD293 cells with liposome and identified with GFP expression and PCR. Myeloid dendritic cells（ DC） were isolated from asthmatic mice during pregnancy and cultured in vitro. The infection efficiency of exogenous genes,carried by recombinant adenovirus,was detected in target cells by fluorescence microscopy. The mRNA transcription level of IL-17R-FcγRⅡb gene,and the expression level of its protein,was determined by RT-PCR and Western blot. The effect of this recombinant adenovirus on the surface markers of the DC,its role to stimulate the proliferation of T cells,and its impact on the secretion of IFN-γ,IL-4 and IL-17,was analyzed by Flow cytometry（ FCM）,CCK-8 method and ELISA method.Results： It was confirmed that the sequence of our vectors and insert fragments were completely correct. The expression of GFP and the PCR results confirmed that our recombinant adenovirus,carrying the target gene,was successfully packaged. We obtained CD11c DC and the infection efficiency of the adenovirus was 85%. At the mRNA and protein levels,the positive transcription and expression of IL-17R-FcγRⅡb can be amplified in line with expectations. No significant difference was funded in the expression of positive CD11c cells among all the three cell groups. Compared with that in the control group and in the EGFP-DCs group,the expression CD80marker increased in IL-17R-FcγRⅡb-DCs group,while the expression of CD86marker decreased（ P 0. 05）. In IL-17R-FcγR Ⅱ b-DCs group,the ability of DC cells to stimulate the proliferation of T cell is much lower than the DC cells in the other two groups at the same concentration ratio（ P 0. 05）. In the supernatant of co-cultured transfection vectors,the levels of IL-4 and IL-17（ Th2 / Th17-related cytokine） decreased,the level of IFN-γ（ Th1-related cytokine） increased（ P 0. 05）,while the ratio of IFN-γ / IL-4 / IL-17 increased respectively. Conclusion： IL-17R-FcγRⅡb fusion gene can induce immune tolerance state of DC,inhibit the proliferation of antigenspecific T cells and affect the differentiation of Th1 / Th2 / Th17 subsets by blocking CD86/ CD28co-stimulatory pathway in vitro. It may play an important role on the formation of immune tolerance defects of asthma during pregnancy,so as on the mitigation of airway inflammation＇s reactions.