小鼠肝脏黏膜相关恒定T细胞在2型糖尿病发病中的作用研究

The role of mouse hepatic mucosal-associated invariant T cells in the development of type 2 diabetes mellitus

目的探讨小鼠肝脏黏膜相关恒定T(MAIT)细胞在2型糖尿病(T2DM)发病中的作用。方法将5周龄雄性C57BL/6小鼠分为模型组与正常对照组,每组各5只,模型组给予高脂饮食16周建立T2DM小鼠模型。利用密度梯度离心分离肝脏单个核细胞,流式细胞仪检测肝脏MAIT细胞比例及干扰素γ(IFN-γ)、肿瘤坏死因子α(TNF-α)、白细胞介素17(IL-17)及IL-4表达水平。分选T2DM小鼠肝脏MAIT细胞与正常小鼠原代肝细胞,共培养后检测肝细胞糖代谢关键酶基因[葡萄糖-6-磷酸酶(G6pc)、丙酮酸激酶M1/2(Pkm)、6-磷酸果糖激酶1(Pfk1)]mRNA的相对表达量。组间比较采用独立样本t检验。结果与正常对照组相比,T2DM小鼠体重显著增加[分别为(49.87±0.99)和(26.41±0.81)g,t=36.76,P<0.05],空腹血糖显著升高[分别为(11.52±0.46)和(6.12±0.48)mmol/L,t=16.22,P<0.05];肝脏脂肪变明显;流式细胞仪检测结果显示肝脏MAIT细胞比例显著升高[分别为(1.06±0.46)%和(0.42±0.20)%,t=2.85,P<0.05],IFN-γ表达显著升高[分别为(27.18±13.14)%和(47.56±13.08)%,t=2.46,P<0.05]。体外共培养实验表明,与正常对照组比较,T2DM小鼠肝脏MAIT细胞正常小鼠原代肝细胞中G6pc表达量升高(t=14.60,P<0.01),Pkm(t=22.27,P<0.01)及Pfk1(t=4.28,P<0.05)表达量降低。结论肝脏MAIT细胞在T2DM中比例升高、IFN-γ表达增加,可能通过促进肝糖输出驱动T2DM发生。

Objective To investigate the role of mouse hepatic mucosal-associated T(MAIT)cells in type 2 diabetes mellitus.Methods Five-week-old male C57BL/6 mice were randomly divided into normal control group(n=5)and model group(n=5).The model of type 2 diabetic mice was established via 16 weeks of high-fat-diet feeding.Hepatic lymphocytes were separated from liver samples using density gradient centrifugation.The frequency of hepatic MAIT cells and cytokine expressions of interferon(IFN)-γ,tumor necrosis factor(TNF)-α,interleukin(IL)-17 and IL-4 in hepatic MAIT cells were analyzed by flow cytometry.The gene expressions of key molecules involved in hepatic glucose metabolism[glucose-6-phosphatase(G6pc),pyruvate kinase M1/2(PKm),6-phosphofructokinase 1(Pfk1)]were evaluated in primary hepatocytes co-cultured with hepatic MAIT cells sorted from diabetic mice.Independent-samples t test was used to compare between two groups.Results Compared with the normal control group,the body weight[(49.87±0.99)vs.(26.41±0.81)g,respectively,t=36.76,P<0.05)and fasting blood glucose[(11.52±0.46)vs.(6.12±0.48)mmol/L,respectively,t=16.22,P<0.05]were increased significantly in the model group.The hepatic steatosis was obvious in the model group.Compared with the normal control group,the frequency of hepatic MAIT cells was increased significantly[(1.06±0.46)%vs.(0.42±0.20)%,respectively,t=2.85,P<0.05]and the expression of IFN-γin hepatic MAIT cells was elevated significantly[(27.18±13.14)%vs.(47.56±13.08)%,respectively,t=2.46,P<0.05]in the model group.In vitro co-culture experiments showed that hepatic MAIT cells from diabetes predominantly upregulated mRNA levels of G6pc(t=14.60,P<0.01)and downregulated mRNA levels of Pkm(t=22.27,P<0.01)and Pfk1(t=4.28,P<0.05)of primary hepatocytes.Conclusions Hepatic MAIT cells of diabetic mouse are elevated in frequency and IFN-γexpression,and contributed to the excessive hepatic output of glucose,which is highly correlated with type 2 diabetes mellitus.