miR-101a在乙型肝炎病毒相关性肝纤维化患者中的表达及对肝星状细胞的影响  

Expression of miR-101a in Patients with Hepatitis B Virus-related Liver Fibrosis and Its Effect on Hepatic Stellate Cells

作　　者：王旭天 孙亮 刘宇方 尚楚智 郑鑫[1] WANG Xu-tian;SUN Liang;LIU Yu-fang;SHANG Chu-zhi;ZHENG Xin(Department of Hepatobiliary Surgery,The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an,Shaanxi,710061,China)

机构地区：[1]西安交通大学第一附属医院肝胆外科,陕西西安710061

出　　处：《现代生物医学进展》2021年第13期2415-2422,共8页Progress in Modern Biomedicine

基　　金：中国红十字基金会INTACT科研基金(XM_HR_INTACT_2020_10_01)。

摘　　要：目的:探究mi R-101a在乙型肝炎病毒(HBV)相关性肝纤维化患者中的表达及对肝星状细胞(HSC)的影响。方法:根据肝纤维化程度将HBV相关性肝纤维化患者进行分组(S0组、S1组、S2组、S3组和S4组),健康受试者作为健康对照组。通过RT-PCR检测肺组织中mi R-101a的表达,并分析mi R-101a与疾病严重程度的关系。使用重组人TGF-β1处理人肝星状细胞系LX-2,并对LX-2细胞转染阴性对照mi RNA模拟物(NC-mimic组)、mi R-101a模拟物(mi R-101a-mimic组)、阴性对照mi RNA抑制剂(NC-inhibitor组)或mi R-101a抑制剂(mi R-101a-inhibitor组),未转染的细胞作为对照组,然后通过RT-PCR或蛋白质印迹检测激活HSC及ECM产生的关键基因(α-SMA、COL1A1、COL1A2和COL3A1)和蛋白(a-SMA、collagen I和collagen III)的表达水平。将SD大鼠随机分为4组:对照组、CCl4组、Ad-control组和Ad-miR-101a组,对大鼠腹腔注射CCl4(1 m L/kg体重)诱导肝纤维化模型,每周3次,共4周。然后将5×10^(9)感染单位的携带mi R-101a的重组腺病毒(Ad-miR-101a)或对照腺病毒(Ad-control)经尾静脉注射到大鼠中。4周后,通过苏木精和伊红(H&E)和Masson三色染色评估肝脏形态和纤维化,通过免疫组化染色评估肝脏α-SMA、E-cadherin、vimentin、Smad4或p-Smad2/3的表达。结果:与健康受试者相比,HBV相关肝纤维化患者肝组织中mi R-101a的表达水平明显降低,并且mi R-101a的表达水平随着患者的严重程度升高而降低(P<0.05)。与未处理的细胞相比,mi R-101a在TGF-β1处理的LX-2细胞中以浓度和时间依赖性方式显著下降(P<0.05)。与未处理的细胞相比,5 ng/mL TGF-β1处理LX-2细胞中的α-SMA、COL1A1、COL1A2和COL3A1m RNA表达水平及a-SMA、collagen I和collagen III蛋白表达水平均显著升高(P<0.05)。与对照组相比,mi R-101a-mimic组的α-SMA、COL1A1、COL1A2、COL3A1和TGF-β1 m RNA和a-SMA、collagen I、collagen III、TGF-β1、Smad3和p-Smad3蛋白表达均下调(P<0.05)。与对照组相�Objective: To reveal the expression of mi R-101 a in patients with hepatitis B virus(HBV)-related liver fibrosis and its effect on hepatic stellate cells(HSC). Methods: According to the degree of liver fibrosis, patients with HBV-related liver fibrosis were divided into S0 group, S1 group, S2 group, S3 group and S4 group, and healthy subjects were taken as healthy control group. The expression of mi R-101 a in the lung tissue of patients with HBV-related liver fibrosis was detected by RT-PCR, and the relationship between mi R-101 a and disease severity was analyzed. Human hepatic stellate cell line LX-2 was treated with recombinant human TGF-β1 and transfected with negative control mi RNA mimics(NC-mimic group), mi R-101 a mimics(mi R-101 a-mimic group), negative control mi RNA inhibitors(NC-inhibitor group) or mi R-101 a inhibitors(miR-101 a-inhibitor group). Untransfected cells served as control groups. Then the expression levels of key genes(α-SMA, COL1 A1, COL1 A2 and COL3 A1) and proteins(a-SMA, collagen I and collagen III) that activated HSC and ECM were detected by RT-PCR or Western blot. The SD rats were randomly divided into 4 groups: control group, CCl4 group, Ad-control group and Ad-miR-101 a group. Rats were intraperitoneally injected with CCl4(1 m L/kg body weight) to induce liver fibrosis model, 3 times a week for 4 weeks. Then, 5×10^(9) infected units of recombinant adenovirus carrying mi R-101 a(Ad-miR-101 a) or control adenovirus(Ad-control) were injected into rats via tail vein. After 4 weeks, liver morphology and fibrosis were evaluated by hematoxylin and eosin(H & E) and Masson trichrome staining, and liver α-SMA, E-cadherin, vimentin, Smad4 or p-Smad2/3 were evaluated by immunohistochemical staining expression. Results: Compared with healthy subjects, the expression level of mi R-101 a in liver tissues of patients with HBV-related liver fibrosis was significantly decreased, and the expression level of mi R-101 a decreased with the increase of patient severity(P<0.05). Compared with untrea

关 键 词：miR-101a 乙型肝炎病毒 肝纤维化 肝星状细胞 上皮间质转化 

分 类 号：R512.62[医药卫生—内科学] R575.2[医药卫生—临床医学]