调节性T细胞对肝纤维化小鼠肝脏免疫细胞的影响

Effects of regulatory T cells on immune cells in the liver of mice with liver fibrosis

目的观察肝纤维化小鼠肝内调节性T细胞(regulatory T cell,Tregs)对具有杀伤能力的免疫细胞的影响。方法将实验小鼠分为肝纤维化组、肝纤维化+CD25抗体组、对照组3组。实验组用四氯化碳诱导小鼠肝纤维化,4周后给予小鼠腹腔注射纯化的CD25单克隆抗体(PC61培养上清),下调体内Tregs水平。对照组小鼠腹腔注射等体积生理盐水。流式细胞分析检测肝脏Tregs水平以及CD8+T细胞(细胞毒性T细胞表面标志)、Kupffer细胞(F4/80)以及NK细胞的表达。实时荧光定量检测不同亚型的Kupffer细胞及IFN-γ在不同组小鼠肝脏的表达。结果肝纤维化小鼠肝脏Tregs明显高于对照组,注射CD25抗体下调Tregs 50%以上。CD8+T细胞、Kupffer细胞在肝纤维化小鼠肝内表达升高,下调Tregs后均无明显改变;但NK细胞和IFN-γ在肝纤维化小鼠肝内表达明显下降,下调Tregs后表达显著上升,其中M1型Kupffer细胞在肝纤维化组表达略有降低,下调Tregs后表达升高;M2型变化不明显。结论Tregs参与对肝纤维化小鼠肝脏的免疫调控,其中对M1型Kupffer细胞和NK细胞抑制作用明显,对CD8+T细胞抑制作用不显著。

Objective To investigate the regulation of regulatory T cell（Tregs） on immune cells that have killing ability in liver fibrosis by depleting Tregs in mice. Methods Experimental mice were divided into the liver fibrosis group, liver fibrosis＋anti-CD25 group and the control group. Liver fibrosis was induced by CCl4, 4 weeks later, purified CD25 monoclonal antibody（PC61 culture supernatant） were intraperitoneally injected in part of mice to reduce the Tregs level. The same volume of saline was injected to the control mice. Flow cytometry was used to detect the level of liver CD4＋CD25＋T cells, CD8＋T cells, Kupffer cells（KCs） and NK cells. Real-time PCR was used to detected the expression of KCs subtype（M1, i NOS; M2, arginase- 1） and interferon- γ（IFN- γ） in the liver. Results The number of Tregs increased in fibrotic livers and decreased by more than 50% after anti- CD25 m Ab treatment, and Foxp3 m RNA level showed the same change. CD8＋T cells and KCs were both increased in the liver of fibrotic mice, but did not change after Tregs depletion. However, NK cells and IFN-γ levels were significantly decreased in fibrosis and increased after Treg depletion. Furthermore, M1 but not M2 KCs were slightly inhibited in liver fibrosis and enhanced after Tregs depletion. Conclusion Tregs are involved in immune regulation of liver fibrosis primarily by suppressing M1 KCs and NK cells, but not CD8＋T cells.