Transplantation of Reduced-Size Fatty Livers

描述(申请人提供)：由于担心原发性无功能，脂肪肝很少用于部分肝移植。这些研究旨在阐明脂肪部分移植物的机制，并开发预防脂肪部分移植物失败的策略。我们将使用减体积大鼠肝移植来验证脂肪变性和部分肝移植协同增加活性氧和氮物种(ROS和RNS)、抑制线粒体功能和抑制肝再生的独特假设。在目标1中，我们将调查脂肪变性是否会增加部分肝移植后的移植物衰竭。高脂饮食和低胆碱饮食可分别诱发大鼠脂肪肝，并缩小至原大小的30%~50%。我们将评估移植后的存活率、肝功能和组织学作为脂肪变性、移植物体积和冷藏时间的函数。在目标2中，我们将测试假设，即在高脂饮食诱导的脂肪部分移植物中ROS和RNS增加，导致损伤。ROS将通过自旋捕获技术和免疫组织化学方法进行测定，RNS将通过硝基酪氨酸和亚硝酸盐/硝酸盐水平进行评估。我们预计，脂肪移植物的ROS和RNS产量将高于精瘦部分移植物。我们还将使用荧光活体内多光子显微镜来鉴定ROS和RNS的细胞来源。将评估抗氧化剂(Ad-超氧化物歧化酶、N-乙酰半胱氨酸和多酚)和抗硝化治疗(一氧化氮合酶抑制剂)对脂肪部分移植物存活和损伤的影响。在目标3中，将评估脂肪变性和移植物体积对能量供应的影响。将通过检测解偶联蛋白2、线粒体通透性转换和呼吸链活性来阐明能量产生缺陷的机制，并将评估线粒体通透性转换抑制剂以及抗氧化和抗营养治疗对线粒体功能的影响。在目标4中，将评估脂肪变性和移植物体积对肝再生的影响。将评估细胞增殖和调节再生的因素(细胞因子、转录因子、生长因子和细胞周期相关基因)。此外，我们还将研究线粒体功能的保护以及抗氧化和抗硝酸治疗是否能促进肝脏再生。综上所述，我们期望这项工作将为缩小体积脂肪移植失败的机制提供基本的新见解，并开发基于机制的策略，以增加脂肪肝在临床部分肝移植中的使用并改善预后。

DESCRIPTION (provided by applicant): Fatty livers are rarely used for partial liver transplantation due to the fear of primary non-function. These studies are to elucidate mechanisms and to develop strategies to prevent failure of fatty partial grafts. We will use reduced-size rat liver transplantation to test the unique hypotheses that steatosis and partial liver transplantation synergistically increase reactive oxygen and nitrogen species (ROS and RNS), inhibit mitochondrial function and suppress liver regeneration. In Aim 1, we will investigate if steatosis increases graft failure after partial liver transplantation. Fatty livers will be induced by high fat diet and choline-deficient diet, respectively, and reduced to 30% to 50% of original size. We will assess survival, liver function, and histology after transplantation as a function of steatosis, graft volume and cold storage time. In Aim 2, we will test the hypothesis that ROS and RNS are increased in high-fat diet-induced fatty partial grafts, leading to injury. ROS will be determined by the spin-trapping technique and immunohistochemistry for 4-hydroxynonenal and RNS will be assessed as nitrotyrosine and nitrite/nitrate levels. We expect that ROS and RNS production will be higher in fatty than lean partial grafts. We will also identify cellular sources of ROS and RNS using fluorescent intravital multiphoton microscopy. The effects of antioxidants (Ad-superoxide dismutase, N-acetylcysteine and polyphenols) and antinitrative therapies (nitric oxide synthase inhibitors) on survival and injury of fatty partial grafts will be evaluated. In Aim 3, effects of steatosis and graft volume on energy supply will be evaluated. Uncoupling protein 2, mitochondrial permeability transition and respiratory chain activity will be determined to elucidate the mechanisms of defective energy production, and effects of mitochondrial permeability transition inhibitors and antioxidant and antinitrative therapies on mitochondrial function will be evaluated. In Aim 4, the effects of steatosis and graft volume on liver regeneration will be assessed. Cell proliferation and factors regulating regeneration (cytokines, transcription factors, growth factors, and cell cycle related genes) will be evaluated. In addition, we will investigate if protection of mitochondria function and antioxidative and antinitrative therapies improve liver regeneration. Taken together, we expect that this work will provide fundamental new insights into the mechanisms underlying reduced-size fatty graft failure and develop mechanism-based strategies to increase the use and improve the outcome of fatty livers for partial liver transplantation in the clinic.