【作者】 董晓灵；

【导师】 王曙光；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 肝脏移植是各类终末期肝病唯一有效的治疗方法,供肝需求的急剧增加和来源的缺乏严重限制肝移植的发展。近年来活体部分肝移植开展迅速,但临床上仍存在许多问题尚待解决,如移植肝的活性、移植肝大小与受体的匹配、移植术后肝功能不全等。其中移植术后肝功能不全是决定术后治疗效果的一个最重要的因素,除与免疫排斥、营养等因素外,还与肝脏缺血再灌注损伤,肝脏再生能力损害有密切关系。肝脏再生而是由成熟的肝细胞在各种不同的肝损伤刺激下发生基因表达和激素分泌的改变,导致处于G0期的肝细胞重新进入G1期而开始细胞增殖,当肝脏被修复后,这种增殖循环便停止。肝再生是一个复杂的过程,涉及肝细胞和非实质细胞及其分泌的细胞因子,是它们共同作用的结果。肝细胞生长因子(HGF)首先被证实为培养的肝细胞的血源性丝裂原,HGF及其受体是肝细胞增殖的基本因子,被认为是肝再生始动因子,是促使肝细胞由G1期向S期转化的最主要因素。HGF主要是由非实质肝细胞(LSEC,贮脂细胞及Kupffer细胞等)分泌,通过旁分泌、自分泌等途径刺激肝再生。IL-6在部分肝切除术后由肝脏(主要为非实质性细胞)及肠道内产生,并且被证实是肝再生必不可少的介质。部分肝移植术后肝再生更是一个特殊、复杂的细胞增殖过程。一方面由于肝脏体积锐减导致肝细胞大量丢失,启动肝再生过程。另一方面肝脏低温保存-缺血再灌注,导致肝脏内皮细胞的损伤,特别是肝窦内皮细胞(LSEC)。LSEC损伤后细胞水肿或血小板和白细胞粘附到损伤的内皮细胞表面导致肝脏微循环障碍,继而发生肝实质细胞的损伤。LSEC的损伤不仅是导致移植后肝脏功能不全的一个重要因素,而且是影响肝部分移植术后肝再生活性的一个主要原因。因为肝窦内皮细胞是肝脏再生的“感应期器”,在肝脏部分切除术后被激活,通过旁分泌作用分泌HGF、IL-6、TNF-a、NO等细胞因子启动和促经肝脏再生。因此,在肝脏移植术后及时发现内皮细胞的损伤并找到有效的治疗策略并及时处理,对改善肝移植术后肝功能不全和增强肝脏再生活性有非常重要的意义。VEGF是一种具有多种潜能的细胞因子,主要是促进血管内皮的增生,体外实验表明能促进肝窦内皮细胞的增生、抑制其凋亡。在肝部分切除术后能促进肝脏增生。最近研究显示,VEGF能减轻由化学因素诱导的急性肝脏损害,并降低其死亡率。有学者研究报道大鼠肝部分切除术后,给予高压氧治疗,能明显降低术后肝脏损害,并发现一个重要的因素为VEGF分泌增加及肝窦内皮细胞增生。那么VEGF能否改善肝脏部分移植术后肝窦内皮细胞功能?能否降低肝脏缺血再灌注损伤?能否改善术后肝功能,目前该方面的研究很少。PI3K/Akt信号途径在细胞增殖、细胞周期及生存中起到重要作用。Akt酶是PI3K下游直接靶蛋白,由PI3K激活后才发挥其生物学效应。Akt作为细胞生存通路P13 K/Akt的关键分子,在促进细胞生长、增殖,促进细胞运动、侵袭,抑制细胞凋亡、促进血管生成等方面发挥了重要作用。活性的Akt表达能刺激无血清环境中的肝星状细胞的增殖,显性负性的Akt蛋白表达能抑制血小板源性生长因子(PDGF)诱发的肝星状细胞的增殖。Akt能够调节NOS的活性、进而增加N0的合成,抑制低血流量下的血小板聚集并与超氧化物结合,形成无毒代谢产物NO3-;从而使肝细胞避免氧自由基所致损害,保护肝细胞。那么VEGF作为一种人体体内最强的血管生长因,能否活化肝窦内皮细胞内Akt酶,发挥其生物学效应呢?目前未见该方面研究报道。本研究通过建立大鼠减体积肝脏移植模型,观察VEGF对大鼠肝部分移植术后肝功能、肝窦内皮细胞形态及功能、术后肝细胞增殖的影响,探讨VEGF能否改善大鼠肝部分移植术后肝功能、促进肝再生。并通过观察VEGF对术后血清HGF、IL-6浓度、肝窦内皮细胞HGF mRNA表达及Akt酶活化的影响,进一步探讨VEGF改善大鼠部分肝移植术后肝功能、降低缺血再灌注损伤,增强肝再生活性的机制,旨在为临床减体积肝脏移植术后肝功能不全提供新的治疗策略。本实验取得的主要成果如下:1.建立了稳定的大鼠系列减体积肝移植模型,同时确定了大鼠30%肝移植术可作为肝部分移植术后急性肝损伤动物模型。为肝部分移植后急性肝损伤、肝再生的研究提供了实验基础。2.明确了外源性VEGF能明显减轻肝窦内皮细胞的损害,减少炎性细胞的浸润,降低血清HA的浓度,改善大鼠肝脏移植术后肝窦内皮细胞功能,从而降低肝脏缺血再灌注损伤。3.外源性VEGF能显著降低肝部分移植术后血清ALT、AST、TBIL的浓度,改善术后肝功能,提高术后生存率。4.外源性VEGF能促进术后肝细胞PCNA的表达,增强肝脏再生活性,促进肝再生。5. VEGF能增强大鼠肝部分移植术后肝窦内皮细胞活化的Akt激酶的表达,并且促进HGF、IL-6等细胞因子的分泌,提出VEGF可能通过活化肝窦内皮细胞Akt酶,激活术后肝窦内皮细胞分泌HGF、IL-6等细胞因子,从而启动和促进肝脏再生新的观点。更多还原

【Abstract】 Liver transplantation has become the ultimate effective therapia for end stage liver dieases. The deficient supply of donator liver, however, has become the major agent to restric the development of livertransplantation. To meet this problem, the living donor tecthnic, with transplantation of liver segments, have been carried out promptly. But many problems, such as the activity of recipient graft, the matching to recieper and liver function failure after operation, are to be solved. The liver function failure of graft has been a most important agent that decide therapeutic efficacy of liver transplantation, and witch concerned with liver ischemical reperfusion injury and the suffering activity of liver regeneration besides immunization rejection and nutritic agents.Liver regeneration has been attributed to hepatocyte proliferation following different kind of injury, reflecting the priming of G0 cells and shift to G0 to the G1/S, accompanied by changes of gene expression and hormone secretion. Once the renovation completed, the proliferation circulation quit. The procedure of liver is very complic, which include the heaptocytes and nonparenchymal cell and its secretion of a large numberof cytokines. First of all , the growth factors, produced by nonparenchymal cell (LSEC,and Ito’s cell Kupffer cell), is proved to be the mitogen which promtes hepatocyte shift from G1 to S. Il-6 is proved to be a indispensable mediator produced majorly by nonparenchymal cell in liver and intestinal gland。The liver regeneration procedure after partial liver transplantation is more complicate and specialer, on the one hand ,the liver regeneration was prompted by the loss of liver tissue ,on the other hand the liver sindusoial endothelial cells were injuried by liver ischemical reperfusion injury, accompanied with liver microcirculation disturbance and hepatic parenchymal cells injury because of cellularedema and platelet leucocyte adhesion. And the injury of LSEC has been a major agent which effect the activity of liver regeneration, because the LSEC is the“inducer”of liver regeneration, HGF/IL-6 and TNF-a、NO secreated by LSEC after liver Partial excision can priminted and enhance hepatocytes proliferation. It is very important to improve liver function and enhance the activity of liver regeneration after liver transplantation that to find the injury of LSEC and givie effective therapy .Vascular endothelial growth factor(VEGF), a potent angiogenic factor, can improve blood vessel endothelium cells hyperplasy, and it has been proved that can promote sinus hepaticus hyperplasy and suppress its apoptosis vitro. VEGF can enhance liver regeneration after partial hepatectomy. Recently study indicated that VEGF can relieve acute severe liver injury(ALI) induced by chemical factor, VEGF treatment significantly reduced the mortality rate of ALI in the rat, and it may provide a new therapeutic strategy for ALI. It was reported that hyperbaric oxygen treatment significantly reduced the liver function damage,and enhance the proliferation of hepatocytes and LSECs ,meanwhile the hyperbaric oxygen treatment significantly increased the expression of VEGF protein in the regenerating liver .Can VEGF improve LSEC function or decrease liver ischemical reperfusion injury? Up to now there are few reports .The role of the phosphoinositide 3-kinase/Akt-signal pathway is very important on differentiation, migration, proliferation and anti-apoptosis, invasion, Angiogenesis. Akt is the downstream target protein of PI3K,and bring into it’s full biological effect after activated by PI3K.Activated Akt stimulates liver stella cells proliferation under no serum condition. Blockage of the expression of Akt can suppress liver stella cells proliferation evoked by platelet-derived growth factor. Akt can accommodate the activity of nitric oxide synthase, and increase the synthesis of nitric oxide, and inhibit platelet aggregation and its combining with hyperoxide, and prevent hepatocytes from damage caused by oxygen free radical. Can VEGF activate Akt of LSEC? Up to now there are few reports.This study identified that VEGF can improve hepatic function and promote liver regeneration by observation the effects of VEGF on the functional biochemistry, the level of hyaluronic acid , the morphous of LSECs, and the expression index of proliferating cell nuclear antigen of hepatocytes of rat following partial liver transplantation. To explore the mechanism of effects of VEGF on improving Liver function, degrading ischemical reperfusion injury, and enhancing the activity of heaptocytes proliferation, we observed the effects of VEGF on the levels of HGF, Il-6, and the expression of HGF mRNA and Akt of LSEC after rat partial liver transplantation. This may provide a new therapy strategy for hepatic inadequacy after for-small-size partial liver transplantation for clinical.In this project we explore the role of VEGF in partial liver transplantation, the results are followed:1. A stabile animal model of rats serial reduced-size liver transplantation were set up and the effect of ratio of rat liver graft on liver function and survival rate were evaluated. This provides a experiment foundation for exploring liver regeneration after partial liver transplantation2. To identify that exogenous VEGF can improve the LSEC function,low down liver ischemical reperfusion injury, and elevate the survival rate of rats subjected partial liver transplantation. This may provide a new clinical treatment strategy for hepatic inadequacy after reduced-size liver transplantation.3. Our experiment show that VEGF can enhance the secretion of HGF, IL-6 by activated LSEC through Akt phosphorylated pathway and promote liver regeneration after partial liver transplantation.更多还原