【作者】 梁佳玉；

【导师】 杨雁；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 硕士

【摘要】 慢性肝炎是我国的常见病,威胁着很多人的健康,大多数患者会发展成肝纤维化或肝硬化。肝纤维化（Hepatic fibrosis,HF）是多种慢性肝病病情发展的共同病理基础,肝脏纤维组织的过度沉积的结果。近年来研究证实,肝星状细胞（Hepatic stellate cell,HSC）的活化和向肌成纤维细胞（Myofibroblast,MFB）转化,导致大量细胞外基质（Extracellular matrix, ECM）的合成,另一方面细胞外基质的降解减少导致其合成与降解的不平衡,是肝纤维化发生的病理关键。TGF-β₁是肝纤维化过程中发挥重要作用,目前研究认为TGF-β₁信号经由Smad途径转导入细胞核内调节靶基因转录从而发挥其生物学效应。复方黄芪提取物（compound astragalus extract,简称CAE）是本室根据中药现代化组合的复方黄芪有效部位群,研究已证实CAE具有活血化淤、抗炎、抗氧化、免疫调节、抗肝损伤和抗肝纤维化等作用和抑制HSC增殖、胶原合成、促凋亡、抑制NF-κB活性等机制。我们在此基础上,采用免疫吸附和Western印记分析等分子生物学方法,进一步研究CAE抗肝纤维化的作用的细胞和分子信号机制,主要内容概括如下:1. CAE抑制HSC来源的MFB细胞增殖作用利用MFB细胞,采用MTT法,建立了血清和细胞因子（TGF-β₁）体外刺激MFB细胞增殖的模型。研究了CAE体外抑制MFB细胞增殖的量效和时效关系。结果显示:（1）CAE(10、20、40、80、160mg·L^-1)呈浓度和时间依赖性地抑制由10% NBS诱导的MFB细胞增殖。（2）CAE(10、20、40、80、160mg·L^-1)呈浓度依赖性抑制TGF-β₁（9pmol/L）诱导的MFB细胞增殖。提示:CAE体外能明显抑制MFB细胞的活化。2. CAE对TGF-β₁诱导的MFB细胞移行浸润的影响我们通过体外模拟正常Disse间隙的微环境及肝纤维化时的相关改变,采用细胞移行实验方法,观察肝纤维化时致纤维化生长因子TGF-β₁对MFB细胞移行的影响。结果显示:TGF-β₁可促进MFB细胞侵袭能力,而CAE可抑制TGF-β₁诱导的MFB细胞的侵袭能力。提示:肝纤维化时Disse间隙微环境的改变促进了MFB细胞的移行,CAE抑制肝纤维化的作用可能与其抑制MFB细胞移行的作用有关。3. CAE抑制HSC来源的MFB细胞的Smad信号转导采用免疫吸附和Western blot方法,观察CAE对TGF-β₁诱导的MFB细胞内pSmad2C, pSmad2L,pSmad3L及Smad2/3蛋白表达的影响。结果显示:（1）TGF-β₁（0.33、1、3、9pmol/L）呈浓度依赖性刺激MFB细胞内Smad2C,Smad2L,Smad3L磷酸化;TGF-β₁（9pmol/L）在5～30min孵育时间,呈时间依赖性刺激MFB细胞内Smad2C,Smad2L,Smad3L磷酸化。（2）TGF-β（19pmol/L）在30min作用时间内,CAE（10、20、40、80mg?L-1）可呈浓度依赖性地抑制TGF-β₁诱导的Smad2C、Smad2L磷酸化。提示:CAE通过抑制Smad2C、Smad2L蛋白磷酸化,干扰TGF-β₁在MFB细胞胞内的信号转导,发挥其抗肝纤维化的作用。更多还原

【Abstract】 Chronic hepatitis is very common in our country that threatens the health of many people. Most of the patients can develop into hepatic fibrosis or hepatic cirrhosis. Hepatic fibrosis is very common in many kinds of chronic liver disease. Excess production of fibre in liver is one of their characters. Recently years much research has approved activated hepatic stellate cell transdifferentiating into Myofibroblast,which results in synthesis of large quantities of extracellular matrix components,on the other hand,the imbalance of production and assimilation of liver extracellular matrix,is the key pathological evidence. TGF-β₁ plays an important role in liver fibrogenesis. At the present study,TGF-β₁ signals are transduced directly from the cell membrane to the nucleus and regulate transcription by smad pathway,consequently exert diverse biology effects.Compound Astragalus Extract （CAE） is the combination of active compound extract from the Astragalus. Previous study from our laboratory showed that CAE had significantly activating blood to resolve stagnation, anti-inflammatory, anti-oxidative, anti-hepatic injury,anti-hepatofibrotic and immuno-regulated effects.Moreover,it suppressed the proliferation of MFBs, collagen production, NF-κB effects, and induced apoptosis.Based on the previous study in our laboratory, Immunoprecipitation and Western blotting molecular biology methods were used, this article was designed to explore the mechanisms of anti-hepatic fibrosis effects of CAE at cellular and molecular levels. The main contents are divided into the following sections: 1. Inhibitory effects of CAE on MFBsVitro models for proliferation of MFBs stimulated with serum and TGF-β₁ respectively were established,which were measured by MTT.The results of concentration and time-inhibition of CAE on proliferation in vitro showed as the followings:（1）CAE（10、20、40、80、160mg?L-1）inhibited the proliferation of MFBs stimulated by 10% NBS in a dose and time dependent manner. （2） CAE（10、20、40、80、160mg?L-1）inhibited the proliferation of MFBs stimulated by TGF-β₁（9 pmol/L）in a dose dependent manner. The results suggested that CAE may suppress MFBs proliferation in vitro.2. Effect of CAE on migration of MFBs induced by TGF-β₁To investigate the effects of CAE on the migration of MFBs induced by TGF-β₁, vitro models were employed to partially mimic invivo microenvironment of Disse space of normal and liver fibrosis. The effects were observed via cell migration experiments. The results showed that TGF-β₁ could induce the invasive capacity of MFBs ,and CAE could decrease invasive capacity of MFBs induced by TGF-β₁. Research suggested the change of Disse space microenvironment accelerate the migratory of MFBs in hepatic fibrosis. The inhibitory effects of CAE on migration of MFBs may be related to its mechanism of anti-hepatic fibrotic effect.3. The effects of CAE on Smad signal transductionThe effects of CAE on the expression levels of pSmad2C, pSmad2L, pSmad3L and Smad2/3 in MFBs induced by TGF-β₁ were investigated by immunoprecipitation and Western blotting methods.The results shows that Smad2C, Smad2L,Smad3L phosphorylation was not only in a concentration dependent manner in MFBs induced by TGF-β₁（0.33、1、3、9pmol/L）but also in a time dependent manner in MFBs co-incubated with TGF-β₁（9 pmol/L）for 5³0min. （2） CAE （10、20、40、80 mg?L-1） inhibited Smad2C, Smad2L phosphorylation in MFBs induced by TGF-β₁ （ 9 pmol/L,30 min） in a dose dependent manner. The results suggested that CAE could interfere in the intracellular signal transduction of TGF-β₁ in MFBs by inhibiting Smad2/3 phosphorylation, which may be one of the mechanisms of anti-hepatic fibrotic effects of CAE.更多还原