【作者】 谢艳爽；

【导师】 郭顺根；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2005， 硕士

【摘要】 肝纤维化是机体对于慢性肝损伤的一种修复性应答,也是各种慢性肝病向肝硬化发展的必经阶段。目前公认的发病机制主要是在肝脏多种细胞及细胞因子(cytokine, CK)参与下,引起细胞外基质(extracelluar matrix,ECM)的合成与降解失衡,即合成大于降解,导致 ECM 过度沉积所致。 现已证实,肝星形细胞(hepatic stellate cell,HSC)是肝脏产生 ECM 的主要细胞,HSC 活化是肝纤维化发生的中心环节。各种损肝因素导致肝损伤,继而引起肝细胞坏死及炎症刺激,相关细胞分泌多种 CK 激活 HSC,产生大量的 ECM。HSC 的活化受多种 CK 的调控,已知转化生长因子(transforming growth factor ,TGF-β)、血小板衍生生长因子(platelet derived growth factor, PDGF)、胰岛素样生长因子-I(insulin-like growth factor, IGF-I)等均属激活 HSC 和促进肝纤维化的关键 CK。 根据促肝纤维化细胞因子所起主要作用的差异,可将其分为丝裂性 CK 和致纤维化性 CK 两类。目前认为,PDGF 是已知生长因子中对 HSC 作用最强的有丝分裂原;而 TGF-β是最关键的致纤维化因子,其对促使 HSC 转化为肌样成纤维母细胞(Myofibroblasts, MFB)并促进分泌胶原纤维起到主要作用。 在人类,迄今为止共发现TGF-β1～3三种亚型,其中以TGF-β1在体细胞内所占比例最高(>90%),活性最强,故也最为重要。 在肝脏,TGF-β1的细胞来源,以往认为只有非实质细胞如库普弗细胞、肝窦内皮细胞和HSC表达TGF-β1mRNA。 Bissell 等证实肝细胞也表达TGF-β1mRNA。正常肝脏中,库普弗细胞是TGF-β1的主要细胞来源,而HSC的表达量很低,肝细胞及内皮细胞表达也很少。肝纤维化过程中,HSC转化为MFB,成为肝脏TGF-β1的主要来源。 1995 年,Sekelsky 等在果蝇体内首先发现了 Smads 蛋白,随后的大量研究证实,Smads 蛋白可将 TGF-β信号直接由细胞膜激酶受体转导入细胞核内,是目前已知唯一的 TGF-β受体(TβR)胞内激酶底物。 目前已发现有 10 种Smad蛋白,根据Smad蛋白结构和功能特点,将其分为三类: ①受体调节型Smads (receptor-regulated Smads, R-Smads),主要有Smad1、Smad2、Smad3、Smad5、Smad8-10。R-Smads是TβR 复合物的下游信号分子,可分别被特异性活化的TβRⅠ型受体磷酸化,因此其作用是通道限制性的;其中Smad2、3参与TGF-β与激活素的信号转导,Smad1、Smad3、Smad5、Smad8-9参与骨形态发生蛋白(bone morphogenetic proteins, BMPs)信号转导,成纤维细胞生长因子(fibroblast growth factor, FGF)生物学作用的发挥必须有Smad10参与。②共同介质型Smads (common mediator Smads, Co-Smads), Smad4是目前在哺乳动物所发现的唯一一个Co-Smads ,也是肿瘤抑制基因DPC4的产物,它的作用不同于Smad 家族的其他成员,其不仅是TGF-β必需的信号中转分子,而且可与所有活化的受体调节型Smads蛋白结合,作为共同递质(common-mediator)再与磷酸化的受体调节型Smads相结合,形成异低聚体复合物后转运至细胞核中激活转录应答。③抑制型Smads(inhibitory Smads, I-Smads), 主要有Smad6、Smad7, 作为信号转导过程的抑制剂可抑制其他两类Smads,形成控制TGF-β信号转导的负反馈环路。目前认为Smads蛋白家族中参与TGF-β信号转导的是2 复方鳖甲软肝方药物血清干预大鼠肝星形细胞 TGFβ-Smad 信号转导通路机制的研究Smad2、Smad3、Smad4、Smad7。现已证实,TGFβ-Smad 信号转导过程可直接刺激 HSC 表型转化,诱导 HSC 凋亡,诱导基质基因表达,抑制基质降解,并且间接影响肝细胞、库普弗细胞、窦内皮细胞多项正常功能的发挥,起到促进肝纤维化发生发展的作用。本实验选取的复方鳖甲软肝方由鳖甲、党参等十一味中药组成,具有扶正祛邪,活血化瘀的功效。其临床有效率达 81.67%,治愈率达 55.23%,系国家级中药新药(批准文号:国药准字 1999,2-102 号)。实验观察了复方鳖甲软肝方药物血清对HSC内TGF-β1信号转导通路上游最为关键的两个节点包括受体(TβRⅠ)表达水平的调节以及受体后信号转导蛋白(Smad3)表达水平的干预作用及其所导致HSC生物学行为的改变,以探讨复方鳖甲软肝方药物血清干预HSC内TGFβ-Smad信号转导的机制。1. 复方鳖甲软肝方药物血清对大鼠 HSC 增殖细胞核抗原表达影响的实验研究HSC 在肝纤维化的发生、发展中扮演着最为重要的角色,是肝纤维化形成的细胞学基础。研究表明 HSC 活化、增殖并大量生成 ECM 的过程,是肝纤维化的中心环节。HSC 活化的主要特征之一是 HSC 增殖。本实验从 HSC 增殖角度,运用免疫细胞化学和图像分析技术方法观察复方鳖甲软肝方药物血清对离体大鼠 HSC 增殖的影响。结果显示,复方鳖甲软肝方药物血清可明显抑制 HSC 中增殖细胞核抗原(proliferatingcell nuclear antigen, PCNA)的表达,本研究提示复方鳖甲软肝方具有抑制 HSC 增殖的功能,进而起到抑制肝纤维化发生、发展的作用。2. 复方鳖甲软肝方药物血清对大鼠 HSC 内 TβRⅠ表达影响的实验研究TGF-β1必须首先与其受体结合才能发挥其复杂的生物学作用。参与TGF-β1信号转导的受体主要是Ⅰ、Ⅱ型受体(TβRⅠ、Ⅱ)。TβRⅠ活化是TGF-β1信号转导的起点和关键一步。本实验运用免疫细?更多还原

【Abstract】 Hepatic fibrosis is a prosthetic response to chronic liver injury, still a stage of development of kinds of chronic hepatopathy to cirrhosis of liver . The mechanism of hepatic fibrosis is the imbalance of synthesis and degradation of extracellular matrix (ECM) by regulation of many kinds of cells and cytokines. Recently, a lot of data indicate hepatic stellate cells (HSC) is the key cell of formation of ECM and HSC is also the core link of occurrenc of hepatic fibrosis. Various kinds of facts lead to damage of liver, then cause Cellular necrosis of liver and inflammation. Cells which correlated with hepatic fibrosis secrete cytokines to activate HSC to develop mass of ECM, and result in a phenomenon that synthesis of ECM is more than degradation of ECM. Many cytokines regulate activation of HSC. Among these cytokines, transforming growth factor(TGF-β), Platelet derived growth factor (PDGF), insulin-like growth factor-I(IGF-I)are key cytokines. There are five Isoforms TGF-β1~5 express in vertebrate, and TGF-β1~3 express in human and mammal. TGF-βis major cytokine which can stimulate the transdifferentiation of HSC to myofibroblasts(MFB) and promote collagen synthesis. Among them, the ratio of TGF-β1 in somatic cell is the highest(>90%) and the activity of TGF-β1 is most powerful. Transforming growth factor-β1 (TGF-β1) regulates diverse biologic activities including cell growth, cell proliferation, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. TGF-βis believed to be a key mediator of tissuefibrosis as a consequence of ECM accumulation in pathologic states such as systemic sclerosis. TGF-βis known to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. The first intracellular mediator of TGF-βsignalling that had been identified was Drosophila MAD (Mothers against decapentaplegic), which is involved in the signalling cascade of dpp (decapentaplegic, the Drosophila homologue to TGF-β) signalling [Sekelsky, 1995]. The first vertebrate homologue reported was the tumour suppressor DPC4 (Deleted in Pancreatic Carcinoma, locus 4). In TGF-βsignalling the binding of a ligand-dimer leads to the formation of the receptor complex consisting of an oligomer of receptor type I and II. Receptor type II auto-phosphorylates and phosphorylates the receptor type I which then phosphorylates and activates the receptor-regulated Smad transcription factors. These R-Smads form oligomers with the Co-Smads (Common-mediator Smads) and enter the cell nucleus where they regulate transcription. The antagonistic or inhibitory Smads (I-Smads) negatively regulate these pathways by preventing receptor-mediated phosphorylation of the R-Smads or formation of a functional complex between R-Smads and Co-Smads. Intracellular proteins that mediate signalling from receptors for extracellular TGF β-related factors. Smad2 is essential for embryonic mesoderm formation and establishment of anterior-posterior patterning. Smad4 is important in gastrulation. Smad1 and Smad5 are activated (serine/threonine phosphorylated) by BMP receptors, Smad2 and Smad3 by activin and TGF-βreceptors. Smads activated by occupied receptors then form complexes with Smad4/DPC4 and move into the nucleus where they regulate gene expression. At present, many studies indicate tradition Chinese medicine against hepatic fibrosis have better clinical effect and lower side effect. Our study investigated the inhibitory mechanisms of fufangbiejiaruanganfang against hepatic fibrosis. The prescription of fufangbiejiaruanganfang includes biejia; dangshen; et al. In this study, we investigated the effect of fufangbiejiaruanganfang on TGF-βsignaling pathway including the expression of TGF-βtype I receptor and the expression of downstream mediator of TGF-βsignalingpathway Smad3 protein. Through this study, we will get more understanding about the molecular mechanisms of fufangbiejiaruanganfang against hepatic fibrosis.更多还原