【作者】 戚红丹；

【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， 2007， 硕士

【摘要】 目的:本实验采用的源于生物发酵的川芎嗪提取物晶体由上海爱普香料有限公司提供,观察了川芎嗪对CCl4诱导的大鼠肝纤维化Ⅰ、Ⅲ型胶原的干预作用及肝细胞基因谱的影响,以探讨川芎嗪抗肝纤维化的作用机制。方法:1.实验动物健康SD雄性大鼠72只,体重180-220g,购自北京维通利华实验动物技术有限公司。2.动物分组实验动物随机分为12组。对照组:腹腔注射精制橄榄油0.2ml/只,3次/周。模型组:参照并改进Hernandez-Munoz法建立肝纤维化动物模型,腹腔注射0.2mlCCl4（精制橄榄油1:6稀释）/只, 3次/周。药物组:腹腔注射川芎嗪混合液0.2ml/只,3次/周。每组再分为2周、4周、6周、8周四个不同的时相。3.试剂及仪器Van Gieson染色液,PBS缓冲液,胃蛋白酶消化液,兔抗大鼠Ⅰ型胶原单克隆抗体、兔抗大鼠Ⅲ型胶原单克隆抗体和抗原修复液,Power VisionTMTwo-Step Histostaining Reagent （PV-6001）兔二抗试剂盒和DAB试剂盒,IV型胶原酶、TRIzol,杂交芯片,DMEM合成培养基和胎牛血清,DEPC处理的生理盐水,图像分析仪:美国Tractor Northern Co.产品,TN-8502 Image Analysis System,酶标仪,UVP杂交炉,Agilent扫描仪,Agilent 2100 Bioanalyzer,分析软件:Genespring和Cluster,倒置相差显微镜,CO₂培养箱,台式低温离心机,恒温水浴锅,蠕动泵。4.实验步骤4.1复制大鼠肝纤维化模型,制备各组肝组织切片;4.2肝组织进行Van Gieson和H-E染色,显示胶原纤维的增生程度和肝组织病理改变程度;4.3肝组织进行免疫组织化学方法检测Ⅰ、Ⅲ型胶原表达情况,利用图像分析仪分析结果;4.4制备各组肝脏血清,检测ALT、AST,显示肝功的改变和肝细胞坏死程度;4.5分离各组肝细胞,培养贴壁,提取RNA,制备基因芯片,对结果进行分析。结果:1. Van Gieson胶原纤维染色结果显示:模型组肝脏内主要由胶原纤维组成的纤维间隔正在或已经形成,并破坏界板结构,分割、包绕肝小叶,部分标本可见假小叶形成。川芎嗪组胶原纤维增生程度明显较模型组低,未见假小叶形成。2. H-E染色不同时相肝组织病理改变镜下观察可见:正常组肝小叶结构完整,中央静脉周围呈放射状排列的肝细胞索,彼此连接成网状;肝细胞体积较大,呈多边形,界限清楚。模型组肝小叶边界不清,结构紊乱,肝细胞索排列紊乱,门管区纤维组织增生显著排列密集,中央静脉周围纤维组织增生并向肝细胞索间延伸,部分形成假小叶,肝细胞普遍浊肿,空泡变性,部分肝细胞坏死,肝窦变窄,门管区及中央静脉可见炎细胞浸润。药物组肝小叶结构清晰,肝细胞轻度肿胀、变性,少见肝细胞坏死。3.本实验肝功能结果可见造模4周之前ALT呈增高趋势,4周后又趋于下降,AST在造模的整个过程中都呈升高趋势,表明肝脏损伤程度加重;4周、6周和8周时药物组的ALT和AST均比模型组低,表明川芎嗪可明显降低ALT和AST活性,从而保护肝细胞,减轻肝细胞损伤及肝纤维化发展。4.川芎嗪对肝纤维化Ⅰ、Ⅲ型胶原表达的影响本实验采用免疫组织化学和图像分析技术方法,从整体观察川芎嗪对CCl4损伤大鼠肝脏Ⅰ、Ⅲ型胶原表达的影响,结果表明,肝纤维化模型组大鼠肝内有大量Ⅰ、Ⅲ型胶原沉积,而川芎嗪治疗组肝内胶原沉积较模型组少。5.川芎嗪对肝纤维化肝细胞基因谱表达的影响本实验采用基因芯片技术,从离体角度观察肝纤维化肝细胞和药物干预后肝细胞差异表达的基因,这些基因包括原癌和抑癌基因、离子通道和运输蛋白基因、细胞周期相关基因、细胞骨架和运动蛋白基因、凋亡相关蛋白基因、DNA转录和修复相关基因、细胞信号转导蛋白基因及代谢和发育相关基因等,其中与胶原有关的主要是基质金属蛋白酶（MMP）、Ⅲ型胶原（ColⅢ）、瘦素受体（Leptin r）、金属蛋白酶组织抑制因子（TIMP）和转化生长因子（TGF-β）mRNA,而川芎嗪可以下调肝细胞Leptin r、TIMP和TGF-βmRNA的表达,从而减轻肝纤维化时肝脏胶原基因的表达。结论:1.川芎嗪可明显减轻大鼠肝纤维化发展过程中Ⅰ型和Ⅲ型胶原的表达,提示川芎嗪对CCl4诱导的大鼠肝纤维化胶原纤维增生有一定的防治作用。2.川芎嗪对肝细胞胶原基因表达并无明显的直接干预作用,但对与胶原有关的Leptin r、TIMP和TGF-β的基因表达具有下调作用,提示通过下调与胶原有关的基因表达,以减少胶原的增生途径可能是川芎嗪发挥防治肝纤维化作用机制之一。更多还原

【Abstract】 Chronic liver disease is a severe disease to harm mankind health. Hepatic fibrosis is the feature owned by both chronic liver disease and the essential stage that develop into hepatic cirrhosis. Pathologically, it is characteristic of proliferation and deposit of fiber tissue which is mainly collagenⅠandⅢin portal canal area and liver lobule. It is well known that damaging factors bring about liver injury, then cause cellular necrosis of liver and inflammation. Cells, which are correlated with hepatic fibrosis secrete cytokines to activate HSC to develop mass of ECM. Subsequently the synthesis of ECM in liver is more than degradation of it, which leads to the occurrence of liver fibrosis.And it is popularly thought that by virtue of the participation of several kinds of cell and cytokines extracellular matrix deposits excessively. And a lot of study data indicate that the activation of hepatic stellate cell is central tache in the occurrence and development of liver fibrosis. Ever since a long time ago, HSC is the main study object about hepatic fibrosis . With the deep study , we realize the important position of cooperation of liver cells . The cells in liver contain hepatic parenchymal cells and hepatic nonparenchymal cells, hepatic parenchymal cells are hepatocytes, and hepatic nonparenchymal cells include SECs, KCs, HSCs and hepatic lymphocytes etc. Hepatocytes hold 80 percent in livervolume or so, and hepatic nonparenchymal cells only hold 6.5 percent. Hepatocytes complete multitude function of liver. The pathologicalchange of hepatocytes refer to cell injury, apoptosis and canceration. So the pathogenesy , prevention and cure of hepatic fibrosis should not ignore hepatocytes.ECM include collagen, non-collagen glycoprotein, proteoglycan, elastin, matrix metalloproteinases, tissue inhibitor of metalloproteinase, adhesion molecule, growth factor and cytokine . ECM have important effects on frame texture and attachment site, it can control attachment, migration, proliferation, differentiation and gene expression.Collagen is the essential component of ECM . There are twenty-seven species of collagens, and there mainly are collagen typeⅠand collagen typeⅢin liver, the ratio is 1:1. Collagen protein holds 5%-10% in total protein of normal liver, and the ratio is up to 50% when hepatic fibrosis happens. The ratio of collagen typeⅠand collagen typeⅢincrease, and in later period it can increase there times than before. So, collagen typeⅠand collagen typeⅢare the essential components of ECM when hepatic fibrosis happens. Collagen typeⅠis mainly secreted by HSC and produce a marked effect in middle and later period. It can promote the activation and proliferation of HSC, and inhibit the hyperplasia of HC and SEC in order to promote the development of hepatic fibrosis. Collagen typeⅢis a kind of fibroid collagen protein, and it increases mainly in the morning of hepatic fibrosis.When gene chip containing thousands of DNA or RNA sequence, which used as probes , on silica wafer or nylon membrane are hybridized to labeld sample, gene expression, DNA sequencing as well as DNA mutation and polymorphism can be analyzed in large scale. The major advance of this technology, as compared to conventional techniques, results from the small size of the array, which allows for a higher accurate, a quicker process and a higher sensitivity enables the parallel screening of large number of genes simultaneously on one chip and provides the opportunity to use smaller amp liation of startingmaterial. Now, its application in medical science has been expanded to research differential gene expression, discover new gene, diagnose disease and so on. Gene chip has a characteristic of high-flux, high sensitivity and high accuracy rating. It can be quick, all automatic, multiparametered and at equal pace to analyze the poly-gene even the whole-gene. Gene chip technique provides a new method and consideration to illuminate the development mechanism of hepatic fibrosis.Tetramethylpyrazine is the effective constituent of rhizome. Animal experiments and clinical researches all confirm that tetramethylpyrazine can activate blood circulation to dissipate blood stasis, inhibit platelet coacervating, broaden arteriola, improve microcirculation, inhibit xidation, rival Ca2+, and resist fibrosis. Our study deployed the n-atural extractive of rhizome, and investigated the change of collagen and gene spectra of hepatocyte.Ⅰ. Experimental study on the effect of tetramethylpyrazine on the expression of collagen typeⅠand collagen typeⅢin hepatic fibrosisOur study adopted special immunohistochemistry method---Van Gieson staining method to detect the proliferation degree of collagen, and semiquantitatively analyzed the proliferation degree. The result was that the collagen fibrils of hepatic fibrosis largely deposited and the proliferation degree was very high. But tetramethylpyrazine obviously reduced the collagen deposition, so, tetramethylpyrazine obviously reduced the proliferation degree of collagen fibrils in hepatic fibrosis.Collagen typeⅠand collagen typeⅢare the essential components of ECM when hepatic fibrosis happens. So, the kinesis change of collagen typeⅠand collagen typeⅢis the important parameters to judge the degree of hepatic fibrosis. In this experiment, we used immunocytochemical staining and image analysis techniques to investigate the effect of tetramethylpyrazine on the expression of collagen typeⅠand collagen typeⅢ. The result indicated that tetramethylpyrazine can remarkably reduce the collagen deposition and degree of injury.Ⅱ. Experimental study on the effect of tetramethylpyrazine on the expression of hepatocellular gene in hepatic fibrosisIn this experiment, we used gene chip technique to detect the differential expression genes with tetramethylpyrazine intervention. These genes included proto- carcinoma and anti-oncogene, ion channel and transport protein gene, cell cycle gene, cytoskeleton and movement protein gene, apoptosis-associated protein gene, DNA transcription and repair gene, cell receptor gene, cell signaling gene, metabolism and growth gene, protein translation and synthesis gene and or so. Gene related to the collagen are MMP, collagen typeⅢ, leptin r, TIMP and TGF-βmRNA. In this experiment, tetramethylpyrazine can remarkably downregulated the expression of leptin r, TIMP and TGF-βmRNA, so reduced the expression of collagen.In summary, tetramethylpyrazine is an effective medicine against liver fibrosis. According to experiments we have conducted, the major mechanisms of tetramethylpyrazine might be involved in the following several aspects :1. Tetramethylpyrazine significantly reduce the expression of collagen of hepatic tissue and the proliferation degree of collagen fibrils.With immunocytochemical staining method, we find tetramethylpyrazine remarkably reduce the expression of collagen typeⅠand collagen typeⅢ.2. With gene chip technique, we find tetramethylpyrazine has no effect on the collagen gene of hepatocyte, but has effect on the gene which related to the collagen, so tetramethylpyrazine might through regulating the expression of the gene which related to the collagen of hepatocyte, affect the expression of collagen in liver.In a word, through the experimental study, we can conclude that tetramethylpyrazine has a certain effect on preventing and treating hepatic fibrosis.更多还原