【作者】 何勇；

【导师】 窦科峰；

【作者基本信息】 第四军医大学 ， 外科学（肝胆外科学）， 2001， 博士

【摘要】 肝细胞生长因子（hepatocyte growth factor，HGF）最早于肝切除后的残余肝组织中被发现，在90年代初期从大鼠血小板中分离提纯，其活性不具种属特异性，因最初被发现可刺激肝细胞合成DNA而得名。肝细胞生长因子是很强的促肝细胞增殖因子，不仅能刺激肝细胞的再生，促进肝功能恢复，而且可改善肝纤维化，在损伤因子刺激时保护肝细胞。研究证实，HGF是迄今为止发现的最强大的刺激肝细胞分裂增殖的物质。因此有关肝细胞生长因子的研究是近年治疗肝脏疾病的研究热点。 研究表明HGF不仅对原代培养的肝细胞是强促有丝分裂原。而且在动物体内实验中，HGF同样在肝脏再生的过程中发挥着高效的促有丝分裂作用。在肝切除术后，血浆HGF快速升高，肝脏贮脂细胞表达HGFmRNA水平增加，同时其它组织的间质细胞如肺、脾的HGFmRNA表达水平也增加，从而促使肝细胞DNA的合成增加。在肝切除术后，HGF不仅可增强肝脏的再生而且在术后24h内可快速改善高胆红素血症，减轻门脉周围炎症和胆小管阻塞。此外，HGF还有肝细胞保护作用，不仅对体外培养的受四氯化碳等肝毒素损伤的肝细胞有保护作用，而且对实验性的肝功能损伤动物模型也有明显的治疗作用。但HGF主要由肝脏间质细胞产生，肝细胞 第四军医大学博土论文6 一 仅能产生HGF的受体C－met。为研究自分泌内源性HGF对肝细胞生长增殖 及抗损伤能力的影响，我们设计了此实验。 急性肝功能衰竭hCll比liVCr历uW，＊LF ）是一种涉及多器官的临床综 合征，由大面积的肝细胞坏死引起，以很快发展的肝性脑病和严重的肝功 能损害为特点。肝功能衰竭的治疗目前仍是世界上一大医学难题，同时肝 功能衰竭也是危害人类健康的重要原因之一。我国是病毒性肝炎高发区， 每年有数百万的人死于肝功能衰竭。在肝胆外科，肝功能衰竭尤其是急性 肝功能衰竭也并不少见。这通常是肝切除术后常见而严重的并发症，也是 导致术后死亡的主要原因。肝切除术后往往引起肝功能的损害，尤其是广 泛肝切除术后，即使手术经过比较顺利，术后也常有轻微的黄疽、血浆蛋 白质降低、血清转氨酶升高等变化。对某些肝切除的患者，特别是合并明 显肝硬化者，则可能发生肝功能不全或衰竭。 本文旨在探讨HGF基因的自分泌表达对肝细胞生长增殖及抗损伤能力 的影响，以及对急性肝功能衰竭动物模型的作用，为急性肝功能衰竭或其 它肝脏疾病的基因治疗提供实验材料和理论依据。本实验进行了以下几个 方面的研究并获得了相应的实验结果： 1．应用基因克隆重组技术，构建了含人肝细胞主长因子基因和绿色荧光蛋 白基因的真核表达载体 pEGFp－HGF，经酶切鉴定证明载体构建正确。 2 采用脂质体介导的基因转染方法，将含人 HGF CDNA的真核表达载体 pEGFPHGF转入人肝细胞系 QZG细胞中，经原位杂交、免疫组化、 荧光显微镜观察证实HGF己获得表达。 3．将稳定转染了 HGF基因的细胞与未转染的细胞的生物学行为进行了比 较观察：从0＊A、RN A、蛋白质等各水平的检测结果均表明，＊＊F对 培养的正常人肝细胞（QZG细胞）的生长增殖有明显的促进作用。而 且转染HGF基因对四氯化碳损伤的肝细胞有明显的保护作用，不仅可 提高染毒细胞的存活率，还可降低受损细胞ALT、K”漏出。 第四军医大学博士论文7 4．建立了急性肝功能衰竭动物模型，在活体进行基因转染，结果证实，活 体内转染HGF基因，对急性肝功能衰竭有一定的治疗作用，体观在提 高了存活率，肝功能状况有一定的缓解，病理组织学观察也证实，大鼠 肝脏组织学改变明显减轻。 综上所述，本实验证实HGF基因对QZG细胞的生长增殖有明显的促 进作用，而且增强了 QZG细胞的抗损伤能力。进一步应用 HGF基因转染 在急性肝功能衰竭动物模型中进行应用，有一定的治疗作用。更多还原

【Abstract】 Hepatocyte growth factor(HGF) was first identified as the most potent mitogen for hepatocytes in sera from 70% hepatectomized rats by Nakamura et al. HGF is a heterodimer composed of a 69kD a-subunit and a 34kD b-subunit. Recently, HGF has been proved to be the same molecule as scatter factor and tumor cytotoxic factor. Several lines of studies have shown that HGF is the long-sought hepatotrophic factor for liver regeneration. Following the onset of various types of hepatic injuries, HGF messenger RNA expression is rapidly up-regulated in the livers of experimental animals. Serum HGF levels are elevated in patients with various hepatic disorders. Extensive in vivo studies on the efficacy of recombinant HGF on hepatic regeneration revealed that HGF elicits a potent hepatotrophic action. HGF strongly stimulates DNA synthesis of hepatocytes in damaged livers, caused by partial hepatectomy or induced by carbon tetrachloride (CC14) administration. Likewise, HGF suppresses the onset of severe hepatic injuries or maintains the integrity of hepatocytes in the livers of mice with cholestasis induced by a -naphthyl-isothiocyanate; this means that HGF mayhave potent cytoprotective or anti-hepatitis actions, as well as mitogenic action for hepatocytes in vivo.HGF is a kringle-containing polypeptide growth factor that preferentially targets a wide variety of epithelial and endothelial cells, as well as mature hepatocyte. HGF is expressed in mesenchymal or stromal cells and acts as a mesenchymal- (or stromal-) derived paracrine factor for neighboring parenchymal cells. In the liver, HGF is synthesized by nonparenchymal cells and targets both parenchymal hepatocytes and bile duct epithelial cells. In addition to mitogenic activity, HGF enhances the motility of various types of cultured cells as demonstrated by "scatter factor". HGF also plays the role of an epithelial morphogen as it induces branching morphogenesis of renal epithelial cells or nonparenchymal epithelial liver cells in collagen gels. The signal-transducing receptor for HGF is c-met protooncogene product of transmembrane tyrosine kinase.Acute liver failure (ALF) is uncommon but not rare; approximately 200 cases occur annually in the United States, with a mortality approaching 80 percent. Few conditions in medicine are more dramatic or more devastating than ALF. Severe liver-cell dysfunction strikes previously well people suddenly, and many of them die. ALF embraces a number of conditions whose common thread is severe injury of hepatocytes or massive necrosis. Loss of hepatocyte function sets in motion a multiorgan response, and death may occur even when the liver has begun to recover. Hepatic encephalopathy and coagulopathy in the setting of an acute hepatic disease define ALF. The term "fulminant hepatic failure" is generally applied to patients in whom hepatic encephalopathy develops within 8 weeks of the onset of illness, whereas "subfulminant hepatic failure" issued to describe a minority of patientsin whom hepatic encephalopathy develops after a10longer illness, up to 26 weeks in duration. ALF is used as the most suitable umbrella term, since it encompasses all these clinical presentations.On the basis of hepatotrophic and biological activities of HGF to reconstruct tissue structures, as well as its preferential target cell specificity, we asked if HGF might be of potential benefit in treating subjects with acute liver failure. As a result of this study, we now report evidence that exogenously injected recombinant HGF prevents in rats the progress of acute liver failure in experimental models.1. Using technique of recombinant DNA, the eukaryotic expression vectors pEGFP-HGF was successful construted. It contained the full-length human HGF cDNA (2.3kb).2. Hepatocyte cell QZG was transfected with pEGFP mediated by lipofect-AMINE. After G418 selection, we obtained successfully transcripted HGF gene of cells. We named it QZG/pEGFP-HGF. The change of the proliferation behaviors was investigated in QZG/pEGFP-HGF. Compared wit更多还原