【作者】 董玲；

【导师】 王吉耀； 姜林娣；

【作者基本信息】 复旦大学 ， 内科学， 2005， 博士

【摘要】 背景：肝纤维化是多种慢性肝病的共同病理基础。肝纤维化发展的各个时期，肝星状细胞的活化和细胞外基质合成与降解的失衡是重要病理机制。其中转化生长因子β₁（TGF-β₁）起了关键作用。TGF-β信号通过跨膜丝氨酸／苏氨酸受体激酶传递入核，胞浆内进化保守的smad蛋白家族起了重要的转导作用。它们主要分为3类：受体调控型smads（R-smads）可直接与活化的Ⅰ型受体结合而磷酸化；共同介导型smads（Co-smads）是必需的中转分子，当R-smads磷酸化并与之形成寡聚体后可转位入核调控靶基因转录；抑制型smads（Ⅰ-smads）是TGF-β信号通路的负反馈调节。其中Smad2、3、4、7参与了信号转导。甘草酸是目前广泛应用于临床的有效的慢性肝病治疗药物。它可能通过诱导干扰素α，抑制白介素6和肿瘤坏死因子表达，诱生前列腺素E，抑制磷脂酶A2的活性而起作用的。既往研究提示甘草酸可能通过干预TGF-B／smad信号通路来发挥抗纤维化作用的，但具体机制尚待进一步明确。目的：研究甘草酸对TGF-β₁刺激的大鼠肝星状细胞中TGF-β／smad信号通路的影响。以期揭示甘草酸抗纤维化的分子机制。方法：用SD大鼠分离肝星状细胞并体外培养。实验分成对照组和不同浓度甘草酸给药组（1μmol／l-1000μmol／l）。用MTT法研究对细胞增殖的抑制，台盼蓝拒染法和上清液LDH测定来研究对细胞活力的影响。ELISA法测定上清中HSC分泌的TGF-β₁。Western blot法检测α-SMA蛋白质表达水平。采用GEArray基因芯片检测对照组，TGF-β₁组，TGF-β₁+甘草酸100μmol／L组的基因表达，筛选与TGF-β／smad通路相关的有变化意义的靶基因。然后分成6组，分别为对照组、TGF-β₁组、TGF-β₁+不同浓度甘草酸组（1μmol／l-1000μmol／l）。采用半定量RT-PCR法检测smad2、smad3、smad7、α2Ⅰ型前胶原、α1Ⅲ型前胶原、纤溶酶原激活物抑制因子-1、TGFβ2型受体mRNA水平，采用Western blot法检测smad2、磷酸化-smad2、smad3、胞核-smad3、smad7、Ⅰ型胶原、Ⅲ型胶原、纤溶酶原激活物抑制因子-1和TGFβ2型受体蛋白质表达水平。结果：MTT实验显示100μmol／L-1000μmol／L甘草酸能明显抑制肝星状细胞增殖但不同浓度甘草酸并不影响肝星状细胞的活力。不同浓度甘草酸能逐渐抑制α-SMA蛋白质表达水平。1000μmol／L甘草酸能抑制上清液中肝星状细胞分泌的TGF-β1。基因芯片结果表明：经TGFβ₁作用后表达上调，再经甘草酸作用后表达下调的基因有16项；经TGFβ₁作用后表达下调，再经甘草酸作用后表达上调的基因有5项；经TGFβ₁作用后表达上调，再经甘草酸作用后表达上调更加明显的基因有2项。其中有7项基因与TGF-β／smad通路相关。RTR-PCR和Western blot结果验证了与基因芯片的一致性。表明TGF-β₁能增加smad2、smad3、smad7、α2Ⅰ型前胶原、α1Ⅲ型前胶原、纤溶酶原激活物抑制因子-1、TGFβ2型受体mRNA表达水平，同样增加smad2，磷酸化-smad2、smad3、胞核-smad3、smad7、Ⅰ型胶原、Ⅲ型胶原、纤溶酶原激活物抑制因子-1、TGFβ2型受体蛋白质表达。1μmol／l-1000μmol／l甘草酸能逐渐降低smad2、smad3、smad7、Ⅰ型胶原、Ⅲ型胶原、纤溶酶原激活物抑制因子-1的mRNA和蛋白质表达水平，但增加TGFβ2型受体的mRNA水平和蛋白质表达。结论：高浓度甘草酸能抑制肝星状细胞增殖和TGF-β₁分泌：不同浓度的甘草酸能减少smad2、smad3、smad7、Ⅰ型胶原、Ⅲ型胶原、纤溶酶原激活物抑制因子-1的mRNA和蛋白质表达水平，但上调TGFβ2型受体mRNA和蛋白质表达水平更多还原

【Abstract】 Background: Liver fibrosis is the common pathological basis for diverse liver injuries. During the formation of liver fibrosis or cirrhosis, the activation of hepatic stellate cells （HSC） and the imbalance between synthesis and degradation of extracellular matrix （ECM） are important in the pathologenesis. Molecular mechanisms involved in fibrogenesis reveal that transforming growth factor β （TGF-β ）, especially TGF- β 1, plays a pivotal role. The signal transduction of TGF-β through a family of transmemberane ser/thr kinase receptors to the nucleus is predominantly mediated by phosphorylation of evolutionarily conserved cytoplasmic mediators of the smad family. The smad family is divided into three functional groups: R-smads （receptor-associated smads） can directly interact with activated type 1 receptor and then be phosphorylated; Co-smads（Common mediated smads） is a common necessary mediator, upon R-smads phospharylation and oligomerization with Co-smads, they move into nucleus to regulate transcription of target genes; and I-smads （inhibitory smads） may function as a negative feedback loop in TGF- β signaling pathway. Smad2、 3、 4 and 7 are involved in TGF-β signaling. Glycyrrhizin is a widely used and effective agent in current clinic treatment for chronic hepatitis. Its effect might be associated with inducing α -Interferon, reducing the expression levels of interlukin 6 and tumor necrosis factor, producing prostaglandin E, and inhibiting the activities of Phospholipase A2. Previous research showed glycyrrhizin may inhibit fibrosis by intervention of TGF β /smad signal. However the accurate mechanism of glycyrrhizin still needs to be investigated. Objective: To investigation the effects of glycyrrhizin on TGF- β /smad pathway in cultured HSC stimulated by TGF- β₁ and then to reveal the possible molecular mechanism of glycyrrhizin on anti-fibrosis..Methods: Primary HSCs were isolated from SD rats and cultured in vitro. The inhibitory rate of proliferation was measured by MTT method, the viability of cells was tested by evaluation of supernant LDH and Trypan blue dye exclusion within control and of glycyrrhizin 4 groups （in different concentration of 1 μ mol/L-1000 μ mol/L）. Protein expression of α -SMA was detected by western blot. TGF- β₁ concentration was also detected by ELISA. The gene expression profiles were compared within control group, TGF- β₁ group or TGF- β₁ with glycyrrhizin （100 μmol/L） group. Using a cDNA microarray GEArray^?Q to screen changed target genes with significant change in TGF-β /smad pathway. Then HSCs were divided into 6 groups, as control group, TGF β₁ only group and TGF β₁ with different concentration glycyrrhizin（1 μ mol/L-1000 μ mol/L） groups respectively. The mRNA levels of smad2、 smad3、 smad7、 procollagen I α 2、 procollagen III α1、 PAI-1、 TGF β receptor 2 were assessed by semi-quantitative RT-PCR method , The protein level of smad2、 p-smad2、 smad3、 N-smad3、 smad7、 type I Collagen、 type III Collagen、 PAI-1 and TGF β receptor 2 were assessed by Western blot analysis. Results: MTT method showed an obvious inhibition of HSC proliferation after 100 μ mol/L -1000 μ mol/L glycyrrhizin treatment. Different concentrations of glycyrrhizin do not affect the vitality on HSC by LDH measurement and Trypan Blue dye exclusion method. Glycyrrhizin of different concentration could gradually decrease protein expression of α-SMA. Glycyrrhizin of 1000 μ mol/L could inhibit supernant TGF- β₁ secreted by HSC. The cDNA microarray identified that 16 genes were down-regulated by glycyrrhizin after being up-regulated by TGF β₁; 5 genes were up-regulated by glycyrrhizin after being down-regulated by TGF β₁; and 2 genes were up-regulated predominantly by glycyrrhizin after being up-regulated by TGF β₁ in HSC. Among these 7 genes were associated with TGF- β /smad signaling. The results of RT-PCR and Western blot identified the coincidence with those of cDNA microarray. It also demonstrated that TGF β₁ could increase mRNA level of Smad2、 3、 7、 procollagen I α 2、 procollagen III α 1, PAI-1 in HSC, and TGF-β₁ could also increase protein expression of Smad2、 3、 7、 p-smad2、 N-smad3、 type I collagen、 type III collagen PAI-1 and TGF-13 receptor 2. Glycyrrhizin of 1 μ mol/1-1000 μ mol/1 could gradually decrease the mRNA level and protein expression of Smad2、 3、 7、 type I collagen、 type III collagen and PAI-1, but increase those of TGF-β receptor 2.Conclusion: Glycyrrhizin with high concentration could inhibit HSC proliferation and TGF β₁ secretion. Glycyrrhizin in different concentrations could also decrease the mRNA level and protein expression of smd2、 smad3、 smad7、 type I 、 type III collagen and PAI-1 gradually. It could also up-graduate the mRNA level and protein expression of TGF- β receptor 2.更多还原