细粒棘球蚴感染小鼠肝精氨酸酶的表达和功能研究

目的分析精氨酸酶(ARG)在细粒棘球蚴感染小鼠肝白细胞中的表达和功能。方法将18只雌性BALB/c小鼠随机分为感染组和对照组,每组9只。感染组小鼠腹腔注射细粒棘球蚴原头节2000个/鼠,对照组注射等量生理盐水。感染后270 d,分离小鼠肝组织中白细胞。蛋白质印迹(Western blotting)分析肝白细胞中ARG和一氧化氮合酶(NOS)的表达情况,采用Image J软件分析蛋白质表达丰度。ARG活性检测试剂盒、尿素检测试剂盒和一氧化氮(NO)检测试剂盒检测肝中白细胞的ARG活性、肝组织的尿素和NO含量。流式细胞术分析肝多种髓系细胞中ARG-1和T细胞中CD3ζ的表达情况。采用SPSS 20.0统计学软件进行数据分析。结果Western blotting检测结果显示,感染组小鼠肝白细胞中ARG-1表达的灰度值为1.03±0.01,高于对照组的0.12±0.01(P<0.01);感染组小鼠肝白细胞中ARG-2和诱导型一氧化氮合酶(i NOS)表达的灰度值分别为0.54±0.01和0.64±0.02,对照组分别为0.55±0.02和0.59±0.02,差异均无统计学意义(P>0.05)。感染组小鼠肝白细胞中的ARG活性、肝组织中的尿素和NO含量分别为(0.03±0.00)U/ml、(0.66±0.02)mmol/L和(14.63±1.55)μmol/L,对照组分别为(0.02±0.00)U/ml、(0.04±0.01)mmol/L和(29.22±0.36)μmol/L,两组差异均有统计学意义(P<0.05)。流式细胞术分析发现,ARG-1在感染组小鼠肝白细胞中CD11b^+CD11c^+、CD11b^+F4/80^+、CD11b^+Gr-1^+、CD11b^+Gr-1^+Ly-6C-Ly-6G^+、CD11b^+Gr-1^+Ly-6C^+-Ly-6G^-和CD11b^+Ly-6G^+中表达的相对平均荧光密度分别为1.41±0.12、1.21±0.06、1.52±0.16、1.30±0.03、1.58±0.12和1.21±0.04,均高于对照组的1.00±0.14、1.00±0.05、1.00±0.01、1.00±0.07、1.00±0.03和1.00±0.02(P<0.05)。CD3ζ在感染组小鼠肝CD4^+和CD8^+T细胞中表达的相对平均荧光密度分别为0.82±0.04和0.78±0.05,均低于对照组的1.00±0.05和1.00±0.07(P<0.05)。结论细粒棘球蚴感染小鼠肝白细胞中ARG-1表达增多,一定程度上拮抗NOS;肝CD4^+和CD8^+T细胞中CD3ζ表达下调。

Expression and function of arginase in livers of mice infected with Echinococcus granulosus

Objective To investigate the expression and function of arginase(ARG)in livers of mice infected with Echinococcus granulosus.Methods Eighteen female BALB/c mice were randomly divided into the infection and control group,9 mice each.Mice in the infection group were each given 2000 live protoscoleces of E.granulosus by intraperitoneal injection,while the control group was injected with the same volume of saline.Liver leukocytes were isolated 270 days after infection.The expression levels of ARG and nitric oxide synthase(NOS)were examined by Western blotting,and the protein abundance was analyzed with the Image J software.The ARG activity in liver leukocytes,and urea and nitric oxide contents in liver tissues were assessed with the ARG activity detection kit,urea detection kit and nitric oxide(NO)detection kit,respectively.The expression of ARG-1 in various myeloid cells and CD3ζin T cells in the liver were analyzed by flow cytometry.Data were analyzed using the SPSS 20.0 software.Results Western blotting showed that the gray value of ARG-1 expression in liver leukocytes in t he infection group was 1.03±0.01,which was significantly higher than that in the control group(0.12±0.01,P<0.01);the expression gray value of ARG-2 and inducible nitric oxide synthase(i NOS)in the infection group was 0.54±0.01 and 0.64±0.02,while that of control group was 0.55±0.02 and 0.59±0.02,respectively;no significant differences were found between the groups(P>0.05).In the infection group,the ARG activity in liver leukocytes,and urea and NO contents in liver tissues was(0.03±0.00)U/ml,(0.66±0.02)mmol/L and(14.63±1.55)μmol/L,respectively,being significantly different from those in the control group(0.02±0.00)U/L,(0.04±0.01)mmol/L,and(29.22±0.36)μmol/L,respectively](P<0.05).Flow cytometry showed that the relative fluorescence density of ARG-1 expression in CD11 b^+CD11 c^+,CD11 b^+F4/80^+,CD11 b^+Gr-1^+,CD11 b^+Gr-1^+Ly-6 C-Ly-6 G^+,CD11 b^+Gr-1^+Ly-6 C^+-Ly-6 G^-and CD11 b^+Ly-6 G^+liver cells in the infection group were 1.41±0.12,1.21±0.06,1.52±0.16,1.30±0.03,1.58±0.12 and 1.21±0.04,r espectively,all items being significantly higher than those in the control group(1.00±0.14,1.00±0.05,1.00±0.01,1.00±0.07,1.00±0.03 and 1.00±0.02;P<0.05).The relative mean fluorescence density of CD3ζin liver CD4^+and CD8^+T cells in the infection group were 0.82±0.04 and 0.78±0.05,respectively,both significantly lower than those in the control group(1.00±0.05,1.00±0.07;P<0.05).Conclusion Higher expression of ARG-1 in liver leukocytes was found in mice infected with E.granulosus,which may antagonize the NOS to some extent;meanwhile,the expression of CD3ζin the CD4^+and the CD8^+T cells was down-regulated.