Tim-3对巨噬细胞功能的调节作用研究

Tim-3 in regulating the activity and phenotype of macrophage

目的探讨Tim-3对巨噬细胞功能的调节作用及其机制。方法通过RNA干扰降低Tim-3在巨噬细胞表面的表达,构建一种稳定低表达Tim-3的RAW264.7巨噬细胞系;以野生型及低表达Tim-3的巨噬细胞为研究对象,探讨Tim-3对巨噬细胞活性及表型的影响。结果将含Tim-3 siRNA及NC对照序列的载体经脂质体分别转染细胞系RAW264.7,以G418加压筛选,获得低表达Tim-3的巨噬细胞稳定株。功能实验显示,激活Tim-3通路促进了巨噬细胞分泌TNF-α、IL-6。同时发现Tim-3通路可以影响CD80/CD86共刺激分子在巨噬细胞表面的表达,并进而影响巨噬细胞的抗原呈递功能,促进Th1、Th17细胞的极化。结论成功构建了重组载体Tim3 siRNA,建立了稳定低表达Tim-3的巨噬细胞系,初步研究结果提示Tim-3信号通路在促进TNF-α、IL-6分泌及调节巨噬细胞共刺激分子表达,促进Th1、Th17细胞分化中发挥重要作用。

Objective To investigate the role of Tim-3 in regulating the activity and phenotype of macrophage.Methods RNA interference（RNAi） technique was used to knock down the Tim-3 expression in macrophage cell line RAW264.7.The role of Tim-3 in regulating the activity of macrophage was investigated by ELISA and flow cytometry respectively.Results Recombinant Tim-3 siRNA vector and control vector were transfected into RAW264.7 cell lines by Lipofectamine 2000.After being selected by G418,the RAW264.7 cell lines stably expressing Tim-3 siRNA were successfully established.ELISA data showed that activation of Tim-3 pathway enhanced TNF-α and IL-6 secretion from macrophage.Interference of Tim-3 expression affected the expression of CD80/CD86 on the surface of macrophage and thus promoted Th1,Th17 polarization.Conclusion Functional assays indicate that Tim-3 plays important roles in promoting TNF-α and IL-6 production by macrophage.More importantly,Tim-3 is involved in the regulation of CD80/CD86 expression on macrophage and promotes Th1 and Th17 polarization.