【作者】 黄娟娟；

【导师】 刘世坤；

【作者基本信息】 中南大学 ， 药理学， 2010， 硕士

【摘要】 目的：探讨在肝损伤早期,酒精和HBV协同作用可能的分子机制；同时观察拉米夫定和水飞蓟素对早期肝损伤的保护作用,并从分子水平探讨二者对纤维化通路信号分子的影响。方法：50只HBV转基因小鼠和20只普通小鼠,予以1周的适应性喂养。按以下两种方案进行实验动物分组,分组一：20只转基因小鼠和20只普通小鼠,各随机分为2组,每组10只：普通小鼠对照组和转基因小鼠对照组,以生理盐水灌胃,5 ml/kg,每天1次；普通小鼠酒精组和转基因小鼠酒精组,以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；分组二：除外转基因小鼠对照组和转基因小鼠酒精组,另30只转基因小鼠随机分为3组,每组10只：拉米夫定组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予拉米夫定(100 mg/kg)灌胃,每天1次；水飞蓟素组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予水飞蓟素(100 mg/kg)灌胃,每天1次；拉米夫定+水飞蓟素组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予拉米夫定(100 mg/kg)+水飞蓟素(100 mg/kg)灌胃,每天1次。连续处理10周后,检测各组小鼠血清ALT、AST水平,HBV DNA载量,肝脏病理组织学改变,肝组织TGF-β1、Smad3、Smad7、CTGF mRNA表达水平及TGF-β1、CTGF、α-SMA蛋白表达水平。结果：酒精刺激对普通小鼠肝功能的水平没有影响,但能促进其肝组织中纤维化相关因子TGF-β1、Smad3、Smad7、CTGF、α-SMA的表达。酒精灌胃增加转基因小鼠体内HBV DNA病毒载量,同时引起转基因小鼠肝损伤较普通小鼠明显加重,表现为转基因小鼠血清ALT、AST水平升高,肝细胞病理损伤及肝组织炎症反应程度加重,但纤维化不明显；其肝组织TGF-β1、Smad3、Smad7、CTGF、α-SMA表达增加。拉米夫定、水飞蓟素分别单独使用或二者联用均不能降低酒精刺激的HBV转基因小鼠体内的病毒滴度；水飞蓟素单独使用或与拉米夫定二者联用可降低酒精刺激的转基因小鼠血清ALT、AST水平,但拉米夫定单独使用不能降低小鼠血清ALT、AST水平；拉米夫定、水飞蓟素分别单独使用或二者联用均可减轻酒精刺激的HBV转基因小鼠肝组织病理损伤,使小鼠肝组织内TGFβ1、Smad3、CTGF mRNA及TGF-β1、CTGF、α-SMA蛋白表达减少,同时进一步增加抑制肝纤维化的Smad7 mRNA的表达。结论：1.酒精刺激对普通小鼠肝功能的水平没有影响,但能促进其肝组织中纤维化相关因子的表达。2.酒精引起转基因小鼠肝损伤较普通小鼠明显加重,其肝组织中促纤维化相关因子的表达较普通小鼠增多,表明酒精和HBV对肝损伤有协同作用；其机制与增加TGF-β1、Smad3、CTGF表达,促进HSC活化以及TGF-β1/Smads通路信号分子表达比例失调有关。3.在肝损伤早期,拉米夫定和水飞蓟素可通过调整TGF-β1/Smads信号通路,减少CTGF表达,抑制HSC活化,从而减少ECM的产生和蓄积等机制,阻断早期肝损伤的发生和进展。更多还原

【Abstract】 ObjectiveTo investigate synergistic effects and molecular mechanism of hepatitis B virus (HBV) and alcohol on liver injury in HBV transgenic mice (HBV-Tg mice).Meanwhile,to investigate the protective effect and mechanism of Lamivudine (LMV) and Silymarin (SIL) on liver injury induced by alcohol drinking in HBV-Tg mice.MethodsThe one program of animal grouping:20 HBV-Tg mice and 20 wild-type mice were randomly divided into 4 groups:alcohol-fed Tg mice group (n=10) and alcohol-fed Wt mice group (n=10),were given intragastric administration with alcohol;control Tg mice group (n=10) and control Wt mice group (n=10),were received intragastric administration with saline.The other program of animal grouping:30 HBV-Tg mice were randomly divided into 3 groups:LMV group (n=10), HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with LMV(100 mg/kg);SIL group (n=10),HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with SIL(100 mg/kg);LMV+SIL group (n=10),HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with LMV(100 mg/kg) and SIL(100 mg/kg).All groups were rasied for 10 weeks.The levels of HBV DNA copy number, ALT,AST in serum,the degree of inflammation,the degree of fibrosis,the mRNA expression of TGF-β1,Smad3,Smad7,CTGF and the protein expression of TGF-β1,CTGF,a-SMA in liver tissue were detected.All the staining figures were scanned with electronic computer and all the data were analyzed with SPSS 13.0 software.ResultsThe serum level of HBV DNA copies number,ALT and AST, the mRNA expression of TGF-β1,Smad3,Smad7,CTGF and the protein expression of TGF-β1,CTGF,a-SMA in liver all increased markedly in alcohol-fed Tg mice(vs control Tg mice,P<0.05).Alcohol consumption induced hepatocyte steatosis and hepatic inflammation in alcohol-fed Tg mice,but the change of liver fibrosis was not remarkable.Lamivudine and Silymarin single use respectively or drug combination failed to suppress the increased serum levels of HBV DNA copies number in alcohol-fed Tg mice.In alcohol-fed Tg mice,Silymarine single use or combined with Lamivudine could decrease the increased serum levels of ALT and AST(vs alcohol-fed Tg mice,P<0.05),but Lamivudine single use could not.Lamivudine and Silymarin single use respectively or drug combination could significantly relieve the histological injury and decrease the increased mRNA expression of TGF-β1,Smad3,CTGF and the protein expression of TGF-β1,CTGF,a-SMA mice(vs alcohol-fed Tg mice, P<0.05),but father improve the mRNA expression of Smad7(vs alcohol-fed Tg mice, P<0.05)in the liver.Conclusions1.Alcohol drinking have no effect on liver function, but enhancing the expression of TGF-β1,Smad3,Smad7, CTGF, a-SMA.2.HBV and alcohol have synergistic effects on early liver injury, by enhancing the expression of TGF-β1,Smad3,CTGF, inducing unbalanced expression of Smads and motivating the activation of HSC.3.Lamivudine and Silymarin single use respectively or drug combination could ruduce the serum level of ALT,AST;relieve the histological damage in the liver of alcohol-fed mice.The mechanism may be inhibiting the expression of TGF-β1,Smad3,CTGF,modulating the expression of Smads and suppressing the activation of HSC.更多还原