METTL3对重症支气管哮喘小鼠T细胞分化的影响

Effect of METTL3 on T cell differentiation in mice with severe asthma

目的探讨METTL3在重症支气管哮喘(哮喘)中对T细胞分化的影响。方法本研究为实验研究。30只SPF级雌性C57BL/6J小鼠使用简单随机分组法分为对照组、普通哮喘组和重症哮喘组小鼠模型,每组各10只。观察各组小鼠行为学改变、气道反应性、支气管肺泡灌洗液中白细胞总数、嗜酸性粒细胞和中性粒细胞浸润情况,检测各组IL-4、IL-17蛋白水平和脾源性CD4^(+)T细胞Th17阳性率。检测各组肺组织和脾源性CD4^(+)T细胞METTL3、IL-17蛋白和mRNA表达。检测METTL3过表达和沉默条件下IL-17表达和Th17阳性率。结果与普通哮喘组和对照组相比,重症哮喘组经不同浓度的乙酰甲胆碱刺激后气道阻力均增加,差异均有统计学意义(均P<0.05)。重症哮喘组支气管肺泡灌洗液中白细胞总数、中性粒细胞和嗜酸性粒细胞水平均高于对照组和普通哮喘组,差异均有统计学意义(均P<0.05)。重症哮喘组脾源性Th17细胞阳性率(3.38±0.17)%较普通哮喘组(1.85±0.13)%和对照组(0.42±0.05)%高(均P<0.05)。重症哮喘组肺组织和脾源性CD4^(+)T细胞METTL3蛋白和mRNA的含量均较普通哮喘和对照组高(均P<0.05),其中重症哮喘组、普通哮喘组、对照组肺组织METTL3蛋白相对表达量分别为0.63±0.01、0.31±0.02、0.12±0.01。METTL3过表达组METTL3和IL-17的蛋白、mRNA水平及Th17细胞的阳性率均高于空载转染组,而METTL3沉默组METTL3和IL-17蛋白、mRNA水平及Th17细胞阳性率均低于空载转染组,差异均有统计学意义(均P<0.05)。METTL3沉默组、空载转染组、METTL3过表达组Th17阳性率分别为(0.03±0.01)%、(0.29±0.04)%、(1.30±0.04)%。结论METTL3在重症哮喘小鼠肺组织及脾源性CD4^(+)T细胞中高表达,同时能调控重症哮喘小鼠Th17细胞优势分化。

Objective To investigate the effect of METTL3 on T cell differentiation in severe asthma.Methods This was an experimental study.A total of 30 SPF female C57BL/6 mice were divided by simple random method into control group,conventional asthma group,and severe asthma group,with 10 mice in each group.Behavior,airway reactivity,and infiltration of WBC,eosinophils and neutrophils in BALF were observed.The protein levels of IL-4 and IL-17 and the positive rate of Th17 in spleen-derived CD4^(+)T cells were detected in 3 groups.The protein and mRNA expression of METTL3 in lung tissue and spleen-derived CD4^(+)T cells were detected.The expression of IL-17 and the positive rate of Th17 were detected under the conditions of METTL3 overexpression and silencing.Results After being stimulated by methacholine of different concentrations,the airway reactivity and inflammatory infiltration of the mice in the severe asthma group were higher than those in the conventional asthma group and control group(P<0.05).The total number of leukocytes,neutrophils,and eosinophils in bronchoalveolar lavage fluid in severe asthma group were significantly higher than those in conventional asthma group and control group(P<0.05).The positive rate of spleen-derived Th17 cells in severe asthma group(3.38±0.17)%were higher than those in conventional asthma group(1.85±0.13)%and control group(0.42±0.05)%(both,P<0.05).The protein and mRNA of METTL3 in lung tissue and spleen-derived CD4^(+)T cells of severe asthma group were higher than those of conventional asthma group and control group(both,P<0.05).The relative expression of METTL3 protein in lung tissue of severe asthma group,conventional asthma group,and control group were 0.63±0.01,0.31±0.02,and 0.12±0.01 respectively.The protein and mRNA of METTL3 and IL-17 and the positive rate of splenic Th17 cells in the overexpression group were higher than those in the empty transfection group(P<0.05);those in the silencing group were lower than those in empty transfection group(P<0.05).The positive rate of Th17 in METTL3 silencing group,in empty transfection group,and in overexpression group were(0.03±0.01)%,(0.29±0.04)%and(1.30±0.04)%respectively(all P<0.05).Conclusions METTL3 was highly expressed in lung tissue and spleen-derived CD4^(+)T cells of severe asthmatic mice,and it can promote the dominant differentiation of Th17 cells.