人胎盘源间充质干细胞通过CD73/Nrf2途径抑制Th1细胞分泌TNF-α减轻急性移植物抗宿主病小鼠肝损伤机制研究  

Mechanism of human placenta-derived mesenchymal stem cells in inhibiting TNF-α secretion in Th1 cells through CD73/Nrf2 pathway to reduce liver injury in mice with graft versus-host disease

作　　者：吴沄桦 张恒超 韩凯悦 赵雅萱 栾希英[1] Wu Yunhua;Zhang Hengchao;Han Kaiyue;Zhao Yaxuan;Luan Xiying(Department of Immunology,Binzhou Medical University,Yantai 264003,China)

机构地区：[1]滨州医学院免疫学教研室,烟台264003

出　　处：《中华微生物学和免疫学杂志》2023年第9期663-670,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(32070781);山东省自然科学基金(ZR2020MH166)。

摘　　要：目的:探究人胎盘源间充质干细胞(human placenta-derived mesenchymal stem cells,hPMSCs)通过CD73/核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)途径抑制CD4^(+)IFN-γ+T(Th1)细胞分泌TNF-α减轻急性移植物抗宿主病(graft versus-host disease,GVHD)小鼠肝损伤的作用机制。方法:流式细胞术分析GVHD小鼠外周血和肝组织中Th1细胞对TNF-α的表达以及CD4^(+)IFN-γ+TNF-α+T(TNF-α+Th1)细胞上PD-1的表达水平。采用苏木精-伊红(hematoxylin-eosin,HE)染色、马松(Masson)染色和免疫荧光染色观察各组GVHD小鼠肝组织病理变化,并进一步用HE染色观察各组GVHD小鼠皮肤和肺组织病理变化。体外建立非条件性外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)向Th1细胞诱导方案,检测TNF-α+Th1细胞比例以及其Nrf2和磷酸化细胞核内核因子-κB(phosphorylated nuclear factor-kappa B,p-NF-κB)的平均荧光强度(mean fluorescence intensity,MFI)。结果:与正常对照组相比,GVHD发病高峰组小鼠外周血和肝组织中TNF-α+Th1细胞比例和TNF-α+Th1细胞对PD-1的表达明显升高(P<0.01);hPMSCs干预可降低小鼠外周血和肝组织中TNF-α+Th1细胞比例(P<0.001),促进小鼠外周血和肝组织中TNF-α+Th1细胞对PD-1的表达(P<0.01,P<0.001),然而shCD73对小鼠外周血和肝组织中TNF-α+Th1细胞的干预效果明显减弱(P<0.05,P<0.01)。肝组织病理学分析结果显示,肝脏中TNF-α+Th1细胞比例分别与Suzuki′s评分、胶原面积和α-SMA的MFI呈正相关(P<0.001)。同样,皮肤和肺组织病理学分析结果也显示,外周血中TNF-α+Th1细胞比例分别与皮肤的Cetkovic-Cvrlje评分和肺的Qiao Shukai评分呈正相关(P<0.001)。PBMCs活化实验也显示hPMSCs能下调TNF-α+Th1细胞比例(P<0.01),上调TNF-α+Th1细胞对PD-1的表达(P<0.05);进一步分析发现hPMSCs可增强TNF-α+Th1细胞中Nrf2的MFI(P<0.05),减弱p-NF-κB的MFI(P<0.01)。结论:hPMSCs可能通过CD73/Nrf2途径上调PD1的表达,�Objective To investigate the mechanism of human placenta derived mesenchymal stem cells(hPMSCs)in the inhibition of TNF-αsecretion in CD4^(+)IFN-γ+T cells(Th1)through CD73/nuclear factor-erythroid 2-related factor 2(Nrf2)pathway to reduce liver injury in mice with graft versus-host disease(GVHD).Methods Flow cytometry(FCM)was used to analyze the expression of TNF-αin Th1 cells and the expression of PD-1 on CD4^(+)IFN-γ+TNF-α+T cells(TNF-α+Th1 cells)isolated from peripheral blood and liver tissues of mice with GVHD.Hematoxylin-hosin(HE)staining,Masson staining and immunofluorescence staining were used to observe the pathological changes in liver tissues of GVHD mice in each group.HE staining was also used to observe the pathological changes in skin and lung tissues of GVHD mice.A nonconditional protocol to induce the differentiation of peripheral blood mononuclear cells(PBMCs)into Th1 cells in vitro was established.The proportion of TNF-α+Th1 cells and the mean fluorescence intensity(MFI)of Nrf2 and phosphorylated nuclear factor-kappa B(p-NF-κB)in this T cell subgroup were detected.Results Compared with the normal control group,the proportion of TNF-α+Th1 cells and the expression of PD-1 on this T cells in peripheral blood and liver tissues of mice in the GVHDhigh group increased significantly(P<0.01).The proportion of TNF-α+Th1 cells in peripheral blood and liver tissues decreased after hPMSCs treatment(P<0.001),but the expression of PD-1 on this T cell subset was promoted in peripheral blood and liver tissues(P<0.01,P<0.001).However,the intervention effects of shCD73 on TNF-α+Th1 cells in peripheral blood and liver tissues were significantly weakened(P<0.05,P<0.01).Liver histopathological analysis showed that the proportion of TNF-α+Th1 cells in liver was positively correlated with Suzuki′s score,collagen area and the MFI ofα-SMA(P<0.001).Similarly,histopathological analysis of skin and lung tissues also showed that the proportion of TNF-α+Th1 cells in peripheral blood was positively correlated w

关 键 词：人胎盘源间充质干细胞 TH1细胞 PD-1 CD73 NRF2 NF-κB 

分 类 号：R575[医药卫生—消化系统]