短发夹RNA沉默Tim3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

摘要:

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。

Abstract:

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 Th1 and Th17 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 Th1 and Th17, 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, Th1 cell levels increased and Th17 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.