RSV毛细支气管炎患儿血液CD4~+CD25~+Foxp3~+Treg与CD40/CD40L关系的研究

探讨CD40/CD40L信号通路对不同病原体毛细支气管炎(以下简称:毛支炎)患儿血液CD4~+CD25~+Foxp3~+ Treg的增殖分化及其分泌抑制性细胞因子TGF-β1、IL-10的影响。取呼吸道合胞病毒(RSV)及非RSV感染毛支炎患儿血液并检测CD4~+CD25~+Foxp3~+Treg的百分率;并将血液中分离的单个核细胞(PBMC)接种于24孔板内,加入CD40L McAb进行阻断,作用72h后采用流式细胞仪检测培养板内CD4~+CD25~+Foxp3~+Treg的百分率,激光共聚焦检测CD4~+CD25~+Foxp3~+Treg细胞表面Foxp3的平均密度,酶联免疫吸附法检测培养上清中TGF-β1、IL-10的含量。RSV毛支炎患儿血液中CD4~+CD25~+Foxp3~+Treg百分率显著低于非RSV毛支炎患儿及正常对照组(P<0.05);RSV毛支炎患儿体外培养PBMC中CD4~+CD25~+Foxp3~+Treg百分率均显著低于非RSV毛支炎患儿、正常对照组和抗CD40L McAb组(P<0.05)。毛支炎患儿PBMC经过体外培养72h后,抗CD40L McAb组培养的细胞中CD4~+CD25~+Foxp3~+Treg表面Foxp3的平均密度显著高于RSV毛支炎患儿组(P<0.05)。PBMC培养上清中IL-10和TGF-β1的水平抗CD40L McAb组明显高于非RSV毛支炎组,非RSV毛支炎组明显高于RSV毛支炎组,差异有统计学意义(P<0.05)。RSV毛支炎患儿体内存在严重的CD4~+CD25~+Foxp3~+Treg数量不足;体外用抗CD40L McAb阻断CD40/CD40L通路可促进RSV毛支炎PBMC中CD4~+CD25~+Foxp3~+Treg的增殖。

Study on the relationship between blood CD4~+CD25~+Foxp3~+ Treg and CD40/CD40L in children with RSV induced brochiolitis

To determine the effect of the CD40/CD40L signaling pathway to the proliferation and differentiation of CD4~+ CD25~+ Foxp3~+ Treg and the secretion of inhibitory cytokines,TGF-β1,IL-10 in the blood of the bronchiolitis infected with different pathogens,the blood of the children who developed bronchiolitis which infected or uninfected with RSV were collected and the percentage of the CD4~+ CD25~+ Foxp3~+ Treg was detected by FCM(flow cytometer).Lymphocytes were isolated and seeded in 24 well plate,after 72h-coculture with CD40L monoclonal antibody to block,the percentage of the CD4~+ CD25~+ Foxp3~+ Treg were detected by FCMCflow cytometer),the expression of PE-CY5-Foxp3 was detected by laser scanning cofocal microscopy, the levels of the TGF-β1and IL-10 in supernatant were detected by enzyme-linked immune-sorbent assay.The percentage of CD4~+ CD25~+ Foxp3~+ Treg in blood of the children who were infected with RSV and developed bronchiolitis(RSV infected group) was significantly lower than those in the RSV uninfected group and the control group(P<0.05).In the in vitro culture system,the percentage of CD4~+ CD25~+ Foxp3~+ Treg in RSV infected group was significantly lower than in RSV uninfected group,the control group and the anti-CD40L McAb group(P<0.05).Cocultured with the CD40L monoclonal antibody,72h later,the average optical density of the PE-CY5-Foxp3 in the surface of CD4~+ CD25~+ Foxp3~+ Treg in anti-CD40L McAb group was higher than in the RSV infected group.(P<0.05).The levels of IL-10 and TGF-β1 in the supernatant of the mixed lymphocyte in anti-CD40L McAb group were significantly higher than in the RSV uninfected group.The RSV uninfected group were significantly higher than the RSV uninfected group.The number of CD4~+ CD25~+ Foxp3~+ Treg in the blood of the children who developed bronchiolitis due to RSV infection is lower than that in the normal controls.The in vitro study demonstrated that blocking the CD40/CD40L signaling with anti-CD40L McAb could promote proliferation of CD4~+CD25~+ Foxp3~+ Treg.