活体动物线粒体biogenesis、fission及fusion对肝脏再生中能量供应影响机制的研究

Liver insufficiency and dysfunction frequently occurs after major liver resection (MLR) in surgical treatment of liver malignancy, living liver donation and after transplantation of small-for-size liver grafts (SFSS). Understanding the mechanisms of liver insufficiency and failure after MLR will facilitate the development of effective therapies. Mitochondria are highly dynamic organelles whose number, morphology, distribution, and activity can be regulated by fusion and fission and mitochondrial biogenesis (MB). However, the importance of mitochondrial network integrity was not evaluated in these important functional studies. This study will investigate the role of MB suppression and the change of fusion and fission in liver insufficiency and failure after MLR/SFSS. In addition, this study will address the novel hypothesis that stimulation of MB and adjust fusion and fission will accelerate recovery of liver function following liver insufficiency and failure. This study is innovative in four aspects. First, to invest MB is essential after liver resection to meet elevated metabolic demand, to replace damaged mitochondria following surgery, and to support liver regeneration processes. Suppression of MB would compromise energy production, slow down repair and recovery process, and lead to liver failure. Second, this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and dynamin-related protein1（DRP1） mitochondrial energy metabolism in response to altered energy demand after transplantation of small-for-size liver grafts; and the mechanisms how to regulate the whole procedure. Third, this study will explore the possibility of MB and adjustment of fusion and fission as a novel therapeutic target for liver insufficiency. Thus new, therapeutics that stimulate MB and promote recovery of liver function would advance the field and clinical medicine significantly. Development of similar compounds, would allow the rescue of livers after operation. Finally, Alterations of mitochondrial mass/number, morphological and functional changes in vivo remain unknown. It is essential to evaluate these newly regenerated mitochondria in vivo. In this study, we will use intravital confocal/multiphoton microscopy technology to directly evaluate alterations in mitochondrial abundance and morphology (mitochondrial GFP), mitochondrial polarization status (TMRM) and mitochondrial function (NADH metabolism) in living mice after MLR/SFSS. In combination with analysis of mitochondrial protein content and activity, we will gain a comprehensive understanding of MB processes after operation and fill a critical gap in our knowledge.

线粒体是高度动态的细胞器，根据不同的代谢需求，其可以通过生物再生(biogenesis)、聚变和裂变(fission and fusion)调节数量、形态和活性。在培养细胞和活体动物中线粒体这一变化之间存在很大差异，通过活体动物的研究能更真实地反映线粒体的这一变化过程，但目前开展较少，机制尚不明确。本课题组前期工作（Am J Transplant. 2012，Free Radic Biol Med.2012）通过对活体小鼠直接观察发现：小体积肝移植后超过75%的线粒体出现去极化改变，并引起ATP的供给降低70%以上，再生抑制。我们最新还发现线粒体的生物再生、聚变和裂变过程也发生改变，并推测与上述现象相关，但机制尚不明确。本课题将在上述研究的基础上，通过多光子、共聚焦显微镜及电镜，活体线粒体特殊显影技术，运用多种分子生物学技术，阐明活体动物线粒体的网络动态平衡的机制及对肝脏再生的影响。

背景：线粒体是高度动态的细胞器，而且是细胞中的能量加工厂，根据不同的代谢需求，其可以通过生物再生(biogenesis)、聚变和裂变(fission and fusion)调节数量、形态和活性。我们前期研究发现小鼠小体积肝移植后超过75%的线粒体出现去极化改变，并引起ATP的供给降低70%以上，再生抑制。并且我们研究发现线粒体能量代谢与肝移植的成功率存在极其密切的联系。研究内容：本课题组在前期研究的基础上进一步对线粒体氧化磷酸化、脂肪转化、能量代谢、再生、聚变、裂变、ROS与肝再生的关系，通过多光子、共聚焦显微镜及电镜，活体线粒体特殊显影技术，运用多种分子生物学技术，阐明活体动物线粒体的网络动态平衡的机制及对肝脏再生的影响。重要结果：1.成功构建了C57BL/6J小鼠肝再生模型。2.运用双光子共聚焦显微镜观察活体动物线粒体功能和状态变化。运用线粒体示踪剂（MTR）和Rh123两种不同颜色的实际标记线粒体，观察其极化和去极化的状态，了解线粒体代谢的不同状态。证实了线粒体能量供应在肝再生中起非常重要的作用。3. 检测Mfn1和Mfn2， Fis1和DRP1、HUVEC、Ras、PGC-1α 以及 OPA-1，同时检测Mfn-/-,Fis1-/-和DRP1-/-小鼠，了解线粒体再生、聚变裂变的调控机制融合、裂变明显影响线粒体能量供应。4.调节线粒体融合裂变的相关基因及其通路探究。关键数据：1.构建稳定的70%PHx100%成活率和85%PHx70%成活率的小鼠模型。2.检测了聚变相关蛋白Mfn1/2的下调证明了其与氧气消耗降低ATP生成减少的关系。3.检测裂变相关蛋白Fis1/DRP1过表达线粒体分裂增加。4.同时检测了线粒体內脊蛋白OPA1上调ATP生成降低。5.同时我们探究活性氧自由基（ROS）和MPT抑制线粒体再生从而导致肝脏能量代谢障碍，抑制肝脏功能的恢复的机制。科学意义：1.揭示活体动物线粒体再生、聚变及裂变的变化规律及其对线粒体数量、功能的影响。2.阐明活体动物线粒体再生、功能受到抑制的机理，以及线粒体聚变裂变的调控机制。3.明确促进线粒体再生，调节线粒体聚变、裂变的干预手段，增加能量的供应，促进肝脏再生。