【作者】 萧瑶；

【导师】 宋后燕； 汤其群； 马端； 于敏；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 肝纤维化是多种慢性肝脏疾病的重要病理历程,近年来认为是损伤后的修复反应,主要是由于肝脏细胞外基质(extracellular matrix,ECM)代谢失衡,导致ECM在肝脏中过渡沉积所致。ECM包括胶原、非胶原糖蛋白、蛋白多糖及弹性硬蛋白,其中胶原蛋白是ECM最重要的组成部分。纤溶酶原激活剂包括组织型纤溶酶原激活剂(tissue plasminogenactivator,tPA)和尿激酶纤溶酶原激活剂(urokinase plasminogen activator,uPA),两者同属于丝氨酸蛋白酶家族,均可激活纤溶酶原(plasminogen,Plg)转变为纤溶酶(plasmin)。tPA的主要功能是激活Plg成为plasmin,后者能够降解血栓中的水不溶纤维蛋白,形成水溶性降解片断,使血栓溶解,血管再通,所以tPA用于治疗急性心肌梗塞等血栓疾病。uPA和它的受体uPAR(urokinaseplasminogen activator receptor)与肿瘤细胞的侵袭,转移有密切的关系。纤溶酶除了直接降解纤维蛋白以外,还能激活基质金属蛋白酶家族的成员(matrixmetalloproteinases,MMPs),后者是ECM中主要的蛋白质水解酶,因此,我们认为纤溶酶激活剂可能在肝纤维化中发挥一定的作用。为了研究tPA在肝纤维化发生发展中的作用,我们利用了转基因动物—tPA基因敲除小鼠。我们首先用野生型和tPA基因敲除小鼠通过一定剂量和浓度的四氯化碳腹腔注射,每周两次,连续注射4周,建立小鼠肝纤维化模型,腹腔注射等量橄榄油的野生型和tPA基因敲除小鼠作为相应的对照。4周后,小鼠被处死,用酶谱学的方法研究tPA在各组小鼠肝脏组织中的活性,结果显示,tPA活性在四氯化碳腹腔注射野生型小鼠肝脏组织中大量增高,而tPA基因敲除小鼠肝组织中没有检出tPA活性。tPA活性在四氯化碳诱导后的增高提示tPA与肝纤维化有密切的关系。苏木素—伊红染色(Hematoxylin and Eosin,HE)显示,tPA基因缺失小鼠在造模后的肝组织损伤以及疤痕形成程度比野生型小鼠明显加重,并且有大量的炎症细胞浸润。组织化学研究VG(Van Gieson)染色和羟脯氨酸含量的检测均显示了胶原蛋白在tPA基因敲除小鼠肝脏组织中有大量的沉积,以上结果说明tPA基因敲除小鼠肝纤维化比野生型小鼠明显加重。肝纤维化是ECM在肝脏中大量沉积导致的,为了进一步的研究tPA基因缺失加重肝纤维化的分子机制,我们研究了tPA基因敲除小鼠和野生型小鼠肝脏组织中ECM生成和降解两方面的差异。a-平滑肌肌动蛋白(a-smooth muscle actin,a-SMA)是肝星型细胞向肌成纤维细胞(myofibroblasts,MFs)转化的主要分子标志,后者是生成ECM的主要细胞。MMPs是ECM降解的主要酶类,基质金属蛋白酶抑制剂—1(tissue inhibitor of metalloproteinase-1,TIMP-I)是其重要的抑制剂。我们分别研究了a-SMA,基质金属蛋白酶—13(matrixmetalloproteinase—13,MMP—13),TIMP-1的蛋白表达水平和基质金属蛋白酶—2(matrix metalloproteinase—2,MMP-2),基质金属蛋白酶—9(matrixmetalloproteinase—9,MMP-9)的活性变化,结果显示tPA基因敲除小鼠肝脏组织a-SMA的表达增高,MMP-2和MMP-9的活性下降,同时MMP-13的表达下降,TIMP-1的表达增高,以上的结果说明tPA基因缺失加剧肝纤维化的程度是通过:1.增加肝星型细胞向肌成纤维细胞的转化使ECM大量产生以;2.MMPs和TIMP-1的比例失调使ECM的降解减少。以病毒为载体,外源转入uPA基因可以减轻大鼠肝硬化,显示肝纤维化过程中uPA有重要的作用。为了研究uPA基因在肝纤维化中的作用,我们使用uPA基因缺失的转基因小鼠和野生型小鼠建立肝纤维化模型。比较各组小鼠肝脏实体和病理组织学等变化,结果显示,四氯化碳处理后的uPA基因缺失小鼠相对于野生型小鼠肝脏实体发白,表面不光滑,质地明显变硬。HE染色显示四氯化碳处理后的肝组织损伤程度比野生型小鼠明显加重,肝细胞出现大量的空泡样改变。VG染色和羟脯氨酸含量的检测说明胶原蛋白在uPA基因缺失小鼠肝脏中的含量明显增高。作为肝纤维化ECM的主要成分,Ⅰ型胶原蛋白的实时定量PCR分析说明四氯化碳处理后,uPA基因缺失小鼠Ⅰ型胶原蛋白表达明显较野生型小鼠增高。uPA基因缺失小鼠肝脏组织中a-SMA mRNA和及其编码蛋白质表达均显著增高,TIMP-1 mRNA及其编码蛋白质表达水平在四氯化碳处理后uPA基因缺失小鼠中都明显增高,但MMP-13蛋白质表达水平在uPA缺失小鼠肝脏组织中降低,其mRNA水平却增高了,其原因尚待进一步研究。以上结果说明uPA加剧肝纤维化的程度是由于ECM的生成增加和ECM降解减少。更多还原

【Abstract】 Hepatic fibrosis is a major histological finding of chronic liver disease caused by excess extracellular matrix (ECM) production and reduced ECM degradation. ECM includes collagen, noncollagenous glycoproteins and proteoglycan. Among which, collage is the most important components of ECM.Plasminogen activators include tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Both of them belong to serine protease family. They activate proenzyme plasminogen (Plg) to biologically active plasmin. tPA, as a FDA permitted drug is wildly used in clinical as a thrombolytic agent. The main biological function of tPA is to cleave Plg into plasmin, which degrades fibrin of thrombus. However, uPA, combines its receptor uPAR, is found to play an important role in tumor invasion and metastasis. Recently, it is understood that plasmin not only directly degrades fibrin, but also activate the family of metalloproteinases (MMPs), which are the main enzyme family to degrade ECM. So, we hypothesize that plasminogen activators might play important roles in liver fibrosis.In order to study the function of tPA in the process of liver fibrosis, we used tPA gene knock out C57/bl mice. Firstly, we took injection intraperitoneally 25% carbon tetrachloride (CCM) (dissolved in olive oil) to mice twice per week for 4 weeks and set up liver fibrosis animal model in both wild-type and tPA gene knock out mice. Mice treated with intraperitoneal injection the same dosage of olive oil as the comparable controls. After 4 weeks, the mice were sacrificed. By means of zymography, we found that tPA activity was unregulated markedly in CCM induced wild-type mice liver compared to olive oil administrated mice liver. As expected, there was no tPA activity found in tPA knock out mice. It seems that tPA might plays an important role in liver fibrosis.HE (Hematoxylin and Eosin) staining found that mice lacking tPA developed more severe morphological injury with lots of inflammatory cells existing. VG (Van Gieson) staining and hydroxyproline analysis showed that the deposition of collagen protein was increased in tPA knock out mice liver after after CCl4 administration compared with wild-type counterparts. It was showed that deficiency of tPA gene accelerated mice liver fibrosis.Liver fibrosis is usually to be caused by excess ECM deposition, we analysis both ECM generation and ECM degradation in wild-type mice and tPA knock out mice.α-smooth muscle actin (α-SMA) is the molecular marker for hepatocyte stellate cells (HSCs) transforming to myofibroblasts (MFs), which is the main cell sources of ECM production. On the other hand, MMPs is the main enzymes to degrade ECM. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is the main inhibitor of MMPs. We examined the protein expression of a-SMA, matrix metalloproteinase-13 (MMP-13), TIMP-1 and the protein activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9).α-SMA expression in CC14 administrated tPA knock out mice livers compared with wild-type mice was increased and MMP-2, MMP-9 activities, MMP-13, TIMP-1 expression were found decreased in the liver of CCl4 administrated tPA knock out mice compared with wild-type counterparts. All the results illustrated that deficiency of tPA aggravated liver fibrosis through promoting HSCs activation and inhibiting ECM degradation by decreasing MMP-2, MMP-9 activities and disrupting the balance between MMP-13 and TIMP-1.Recently, many studies showed that overexpression of uPA in the liver may initiate upstream of matrix proteolysis cascade to degrade the deposition of ECM and reverse hepatic fibrogenesis. We used uPA knock out mice model to study the effect of uPA in liver fibrosis. The method used in setting up liver fibrosis mice model is the same as mentioned above. Through observing the mice liver, we found that after CCl4 administration, the surface of uPA knock out mice liver was granulated; the color was much whiter and was harder compared with wild-type counterparts. HE staining showed that mice lacking uPA developed more severe morphological injury and lots of hepatocytes showed vacuole liking changes. VG staining and hydroxyproline analysis displayed an increased deposition of collagen in the uPA knock out mice liver, moreover, the mRNA analysis of collagen I, which is the main ECM gradient in liver fibrosis, showed that collagen mRNA expression increased in uPA knock out mice liver after CCl4 administration compared with wild-type counterparts. The results above illuminated that disruption of uPA accelerated liver fibrosis. Then, we analysisα-SMA mRNA and protein expression, the results showed that deficiency of uPA increasedα-smooth muscle actin both mRNA and protein expression in the mice livers. And deletion of uPA increased TIMP-1 mRNA and protein expression in the mice liver. However, although, uPA null mice showed decreased MMP-13 protein expression, the mRNA expression was higher compared with wild type mice. It needs to be studied further. These results illustrated that deficiency of uPA aggravated liver fibrosis through promoting HSCs activation and inhibiting ECM degradation by promoting TIMP-1 and decreasing MMP-13 expression.更多还原