自身免疫肝炎患者外周血PBMCs自噬基因表达及Th17/CD4^+CD25^+CD127^(low)Treg细胞与肝脏功能相关性研究  

Expression of autophagy gene in PBMCs and correlation between Th17/CD4+CD25+CD127 lowTreg cells and liver function in autoimmune hepatitis

作　　者：汪永强[1] 李显勇[1] 杨琳[1] 刘世军 项明[1] WANG Yong-qiang;LI Xian-yong;YANG Lin(The Second People's Hospital of Neijiang,Neijiang 641001,China)

机构地区：[1]四川省内江市第二人民医院检验科,四川内江641001

出　　处：《中国实验诊断学》2020年第6期934-940,共7页Chinese Journal of Laboratory Diagnosis

基　　金：2017年四川省医学科研青年创新课题(项目编号:Q17051)。

摘　　要：目的通过检测自身免疫性肝炎(AIH)患者外周血单个核细胞(PBMCs)中自噬相关基因Beclin 1、LC3mRNA表达及外周血Th17细胞、调节性CD4^+CD25^+CD127^(low)(Treg)细胞含量,分析与肝脏功能相关性,探讨自噬与免疫平衡在AIH疾病发生中的作用。方法选取经我院诊断为AIH患者35例为AIH组,同时期我院健康管理中心体检者31例为对照组(control组),应用逆转录实时荧光聚合酶联反应RT-qRCR检测两组PBMCs细胞中Beclin1、LC3mRNA表达情况,用间接免疫荧光检测抗核抗体(ANA)、抗平滑肌抗体(ASMA),免疫印迹法检测ANAs以及肝病相关自身抗体:抗肝肾微粒体抗体(LKM)、抗线粒体抗体M2、抗可溶性肝抗原SLA/LP、抗细胞浆肝抗原LC-1、抗核蛋白sp100、抗核蛋白gp210抗体。流式细胞术检测外周血细胞中Th17和Treg细胞在淋巴细胞亚群中相对CD4^+T的相对百分率。全自动生化仪检测肝脏生化指标ALT、AST、GGT、AKP、TBIL、IgG水平,ELISA检测外周血清中白细胞介素IL-17含量。结果 AIH组ALT、AST、TBIL、GGT、AKP、IgG均高于对照组,差异具有统计学意义(P<0.001)。AIH组ANA及免疫性肝病自身抗体阳性率为74.3%、65.7%,均高于对照组,P=0.000。AIH组Beclin 1基因相对表达量为(5.73±4.07)×2^(-7),与对照组比较,差异无统计学意义(t=0.587,P=0.559),AIH组LC3mRNA基因表达相对含量为(2.82±2.06)×2^(-6),对照组为(6.45±5.04)×2^(-6),差异具有统计学意义(t=-3.745,P=0.001)。AIH组Th17/CD4^+T均值为2.61±2.09%,对照组为1.27±0.99%(t=3.394,P=0.001),Treg/C4^+T在AIH组为4.86±2.49%,低于对照组(t=-5.882,P=0.000),AIH组血清IL-17结果为16.03±5.21pg/ml,对照组12.86±4.19pg/ml(t=2.704,P=0.009)。AIH组中LC3mRNA与ALT的相关系数r为-0.360,P<0.05,与AST、AKP、TBIL、GGT、IgG相关系数分别为r=-0.178、-0.166、-0.097、-0.074、0.003,P>0.05。Th17/Treg与ALT、GGT的相关性分别为r=0.596、0.506,P<0.05;与AST、AKP、TBIL、IgG的相关性分别为r=0.260、0.092�Objective This study aimed to analyze the correlation with liver function and explore the role of autophagy and immune balance in the pathogenesis of autoimmune hepatitis(AIH)through the detection of expression of Beclin 1and LC3mRNA in peripheral blood mononuclear cells(PBMCs)and the content of Th17cells and CD4+CD25+CD127lowTreg cells in peripheral blood of patients with AIH.Methods Thirty five patients with AIH diagnosed by our hospital were recruited in this study as the AIH group and 31healthy people in the health management center of our hospital as the control group.The expression of Beclin 1and LC3 mRNA in PBMCs of the two groups was detected by RT-qPCR and ANA and ASMA were detected by indirect immunofluorescence.The anti hepatorenal microsomal antibody(LKM),anti mitochondrial antibody M2,anti soluble liver antigen SLA/LP,anti cytoplasmic liver antigen LC-1,anti nucleoprotein Sp100and anti nucleoprotein gp210were detected by Western blot.The relative percentage of Th17 and Treg cells in CD4+T lymphocyte subsets was detected by flow cytometry.The levels of ALT,AST,GGT,AKP,TBIL and IgG were detected by automatic biochemical instrument,and the IL-17content in peripheral serum was detected by ELISA.Results The index of liver function of ALT,AST,TBIL,GGT,AKP and IgG in AIH group were higher than those in control group,P<0.001.The positive rates of ANA and autoantibodies related with liver disease were74.3%and 65.7%,which were higher than those in control group P=0.000.The relative expression of Beclin 1gene in AIH group was(5.73±4.07)×2-7,which was no statistically significant(t=0.587,P=0.559)compared with control group.However.The relative expression of LC3mRNA in AIH group and control group were(2.82±2.06)×2-6、(6.45±5.04)×2-6,t=-3.745,P=0.001.The mean value of Th17/CD4+T were 2.61±2.09%and 1.27±0.99%in AIH and control group respectively.t=3.394,P=0.001.Treg/C4+T in AIH was 4.86±2.49%.which was lower than that in control group(t=-5.882,P=0.000).And the IL-17level were 16.03±5.21and 12.86±4.19pg/

关 键 词：自身免疫性肝炎 自噬基因 Th17细胞 TREG细胞 肝脏功能 

分 类 号：R446.6[医药卫生—诊断学]