MBD2对Th2/Th17细胞平衡的调控及其在哮喘中的作用研究

Regulation of MBD2 on Th2/Th17 cell balance and its role in asthma

目的:探究甲基CpG结合区域蛋白2(MBD2)在哮喘中的作用及其对Th2/Th17分化的影响。方法:在条件性敲除CD4+T细胞中MBD2表达的小鼠构建以中性粒细胞浸润为主的哮喘模型,并将小鼠分为4组:WT生理盐水组(A组)、WT哮喘组(B组)、CD4CreMBD2fl/fl生理盐水组(C组)、CD4CreMBD2fl/fl哮喘组(D组),每组10只。应用RT-PCR和Western blot实验检测MBD2在各组小鼠脾脏组织中的表达;使用乙酰胆碱(Mch)激发试验检测各组小鼠的气道高反应;收集小鼠肺泡灌洗液(BALF),使用血细胞计数法计算其中的细胞总数、嗜酸性粒细胞和中性粒细胞数量,ELISA法检测细胞因子IL-4与IL-17的表达,HE染色观察各组小鼠肺组织的病理学改变;免疫磁珠分选各组小鼠脾脏中的CD4+T细胞,流式细胞术、RT-PCR及ELISA分别检测Th2、Th17细胞比例及其相关细胞因子的表达水平;分离并培养小鼠na?ve CD4+T细胞,通过慢病毒载体以靶向沉默细胞中MBD2基因表达,流式细胞术检测其对na?ve CD4+T细胞Th2与Th17分化的影响。结果:与A、B组小鼠相比,C组与D组小鼠脾脏组织中MBD2的mRNA及蛋白表达均显著降低(P<0.01);A组与C组小鼠肺组织结构正常,而B组小鼠肺组织中支气管黏膜充血水肿,管腔狭窄,黏膜上皮细胞出现大量坏死脱落,大量炎症细胞浸润,D组介于A组与B组之间;与A组小鼠相比,B组与D组小鼠的气道反应性显著增高(P<0.05),且BALF中细胞总数和嗜酸性粒细胞、中性粒细胞、IL-4及IL-17均显著增加(P<0.05),C组中仅有嗜酸性粒细胞及IL-4明显增加(P<0.01);而与B组相比,D组小鼠中嗜酸性粒细胞及IL-4表达均明显增加(P<0.05)。在脾脏淋巴细胞中,B组与D组小鼠的Th2、Th17细胞比例、GATA3和RORγt mRNA及IL-4与IL-17表达水平均较A、C组小鼠显著增加(P<0.01),与B组相比,D组小鼠中以Th2细胞及相关细胞因子占主导地位(P<0.05);成功利用慢病毒载体沉默小鼠na?ve CD4+T细胞MBD2的表达,且沉默其表达后能显著促进Th2细胞的分化比例(P<0.05),而Th17细胞比例明显降低(P<0.05)。结论:敲除CD4+T细胞中MBD2基因表达可通过促进Th2细胞及IL-4细胞因子表达,而抑制Th17细胞及IL-17表达从而改善以中性粒细胞浸润为主的重症哮喘。

Objective:To explore the role of methyl CpG binding region protein 2(MBD2)and its effect on Th2/Th17 differen‐tiation in asthma.Methods:The asthma model was established in conditional knockout mice with MBD2 expression in CD4+T cells.The mice were divided into four groups:WT saline group(group A),WT asthma group(group B),CD4creMBD2fl/flsaline group(group C),CD4creMBD2fl/flasthma group(group D),with 10 mice in each group.RT-PCR and Western blot were used to detect the ex‐pression of MBD2 in the spleen tissue of each group.The airway hyperresponsiveness of mice in each group was detected by acetylcho‐line(Mch)challenge test.The total number of cells,the number of eosinophils and neutrophils in BALF were calculated by blood cell counting method,the expression of IL-4 and IL-17 was detected by ELISA,and the pathological changes of lung tissue were observed by HE staining.CD4+T cells in spleens of mice in each group were sorted by immunomagnetic beads,and the percentages of Th2 and Th17 cells and the expression levels of related cytokines were detected by flow cytometry,RT-PCR and ELISA,respectively.Mice na‐ive na?ve CD4+T cells were isolated and cultured.MBD2 gene expression in the cells was silenced by lentiviral vector.Flow cytometry was used to detect the effect of MBD2 gene expression on Th2 and Th17 differentiation of na?ve CD4+T cells.Results:Compared with group A and group B,the mRNA and protein expression of MBD2 in spleen tissue of group C and group D were significantly decreased(P<0.01).The lung tissue structure of mice in group A and C was normal,while the lung tissue of mice in group B showed congestion and edema of bronchial mucosa,stenosis of lumen,A large number of necrosis and shedding of mucosal epithelial cells,and a large number of inflammatory cell infiltration.Group D was between groups A and B.Compared with group A,the airway responsiveness of group B and group D were significantly increased(P<0.05),and the total number of cells,eosinophils,neutrophils,IL-4 and IL-17in BALF were significantly increased(P<0.05),only eosinophils and IL-4 in group C were significantly increased(P<0.01).Com‐pared with group B,the expression of eosinophils and IL-4 in group D were significantly increased(P<0.05).In spleen tissue,the per‐centage of Th2,Th17 cells,GATA3 and RORγt mRNA and the expression levels of IL-4 and IL-17 in group B and group D were sig‐nificantly higher than those in group A and group C(P<0.01).Compared with group B,Th2 cells and related cytokines were dominant in group D(P<0.05).MBD2 expression in mouse na?ve CD4+T cells cells was successfully silenced by lentiviral vector,and the differ‐entiation ratio of Th2 cells was significantly improved after silencing the expression of MBD2 cells(P<0.05),while the proportion of Th17 cells was significantly decreased(P<0.05).Conclusion:MBD2 gene knockout of CD4+T cells can improve the expression of Th2 cells and IL-4 cytokines,but inhibit the expression of Th17 cells and IL-17,so as to improve the severe asthma with neutrophil infiltration.