短发夹RNA沉默Tim-3分子表达对哮喘小鼠外周血Th1和Th17细胞的影响

Small hairpin RNA silencing Tim-3 affects peripheral blood Th1 and Th17 cells differentiation in asthmatic mice

目的建立小鼠支气管哮喘模型,采用T细胞免疫球蛋白黏蛋白分子-3(Tim-3)特异短发夹RNA(shRNA)沉默外周血单个核细胞(PBMCs)中Tim-3的表达,探讨Tim-3对辅助性T细胞1(Th1)和Th17细胞分化的影响。方法用卵白蛋白(OVA)致敏并激发建立哮喘小鼠模型,分离哮喘小鼠PBMCs,用Tim-3特异的shRNA片段沉默哮喘小鼠PBMCs中高表达的Tim-3基因。Real-time PCR和Western blot检测Tim-3的表达,流式细胞分析技术(FACS)检测Th1和Th17的比例;ELISA检测细胞培养上清液中干扰素-γ(IFN-γ),白介素-4(IL-4),和白介素-17(IL-17)的水平。结果哮喘组小鼠PBMCs中Tim-3 mRNA表达明显升高;使用特异Tim-3 shRNA沉默哮喘小鼠PBMCs后,沉默组PBMCs中Th1细胞比例显著升高,Th17细胞比例显著下降,与阴性对照组相比差异有统计学意义(P<0.01);沉默组PBMCs培养上清液中IFN-γ明显升高,IL-17明显降低,与阴性对照组相比差异有统计学意义(P<0.05),IL-4水平无明显变化。结论特异Tim-3 shRNA有效地沉默了Tim-3的表达,Tim-3表达的改变影响了T细胞的分化。

Objective To investigate the effects of down-regulating Tim-3 gene in the peripheral blood mononuclear cells（PBMCs） of an asthmatic mouse model by short hairpin RNA（shRNA） and to explore the effect of Tim-3 on Thl and Thl7 cell differentiation. Methods An asthmatic murine model was established by ovalbumin sensitization and challenge. PBMCs were isolated from asthmatic mice and transfected by shRNA targeting Tim-3 gene. The mRNA and protein expressions of Tim-3 were detected by quantitative PCR and Western blot. Flow cytometry analysis was performed to determine the levels of Thl and Thl7, and ELISA was performed to determine concentrations of IFN-γ, IL-4 and IL-17 in the supernatant. Results Tim-3 mRNA expression in PBMCs was significantly increased in asthmatic mice. The mRNA and protein expression of Tim-3 decreased significantly in the shRNA group. Compared with the negative groups, Thl cell levels increased and Thl7 cell levels decreased significantly in the asthmatic groups after Tim-3 shRNA interference. In the Tim-3 shRNA interference groups concentrations of IFN-γ increased significantly while IL-17 decreased significantly. Conclusions Specific Tim-3 shRNA effectively silences the expression of Tim-3 and change in Tim-3 expression could affect T cell differentiation.