ALCOHOLIC HEPATITIS PATHOGENESIS AS DETERMINED FROM HUMAN LIVER TISSUE ANALYSIS

描述(申请人提供)：在肝脏活检组织水平上，对酒精性肝炎的分子病理变化缺乏基本的了解。这是由于技术上的困难，阻碍了对肝活检组织的检查，而不是观察到的形态变化。最近，PI的实验室开发了一些技术，现在可以检测酒精性肝炎患者肝活检组织中基因表达的变化。长期目标是在分子水平上更好地了解基因表达的变化，并将它们与细胞形态水平相关联。这种特殊应用的目的是确定：1)酒精性肝炎细胞周期停滞的机制；2)肝窦内巨噬细胞的病理生理学。 在酒精性肝炎中的作用包括阻断肝窦血流，引起肝细胞缺氧损伤；3)酒精性肝炎肝细胞气球变性的机制。由于激光捕获解剖和对不同细胞群中蛋白质的荧光强度形态测量等新技术的发展，这些问题现在可以在肝脏活检组织上直接得到回答。中心假说是巨噬细胞肝窦阻塞导致细胞周期停滞、球状肝细胞表观遗传转化和小叶中心肝细胞缺氧，共同损伤肝细胞，导致酒精性肝炎功能丧失和肝衰竭。在气球状肝细胞中发展的表观遗传学变化导致癌前肝细胞的形成和肝细胞癌的发展。这一假设是根据申请人实验室提供的初步数据提出的。提出这项研究的理由是，了解酒精性肝炎肝损伤的基本机制将促进治疗酒精性肝炎的新的和更有效的治疗方法，在这种治疗方法中，细胞周期 停滞被逆转到正常；阻塞性巨噬细胞被从血窦中移除，气球细胞退化被防止。在强劲的初步数据的指导下，这一假说将通过追求三个具体目标来检验。1)确定p21诱导细胞周期停滞的机制；2)确定4种类型的巨噬细胞阻断导致小叶中心缺氧性损伤的血窦；3)确定以球状细胞变性为特征的肝细胞表观遗传转化。这种方法是创新的，主要是因为它利用了上述在申请人实验室开发的新技术，首次能够直接研究酒精性肝炎患者的肝脏活检。这项拟议的研究意义重大，因为预计当了解了酒精性肝炎的机制后，将有可能设计出挽救生命的疗法，并节省目前用于治疗酒精性肝炎的主要成本。

DESCRIPTION (provided by applicant): There is a fundamental lack of understanding about the changes in molecular pathology in alcoholic hepatitis at the liver biopsy tissue level. This isa result of technical difficulties which impede the examination of liver biopsy tissue beyond the morphologic changes observed. Recently techniques have been developed in the PI's laboratory, which now make it possible to examine the changes in gene expression in liver biopsies from patients with alcoholic hepatitis. The long term goal is to better understand the changes in gene expression at the molecular level and to correlate them with the cellular morphology level. The objective in this particular application is to determine, 1) the mechanism of cell cycle arrest in alcoholic hepatitis; 2) the pathophysiology of macrophages in the sinusoids in alcoholic hepatitis including their role in obstructing sinusoidal blood flow causing hypoxic injury to the hepatocytes; 3) the mechanism of balloon cell degeneration of hepatocytes in alcoholic hepatitis. These questions can now be answered directly on liver biopsy tissue due to the development of new technologies such as laser capture dissection and fluorescent intensity morphometric measurements of proteins located in the different cell population. The central hypothesis is that cell cycle arrest, epigenetic transformation of balloon hepatocytes and hypoxia of centrilobular hepatocytes caused by sinusoidal obstruction by macrophages, combine to injure hepatocytes, causing loss of function and liver failure in alcoholic hepatitis. The epigenetic changes that develop in ballooned hepatacytes lead to preneoplastic hepatocyte formation and development of hepatocellular carcinoma. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that understanding the fundamental mechanism of liver injury in alcoholic hepatitis will foster new and more effective treatments for alcoholic hepatitis in which cell cycle arrest is reversed to normal; obstructing macrophages are removed from the sinusoids and balloon cell degeneration is prevented. Guided by the strong preliminary data, this hypothesis will be tested by pursuing three specific aims. 1) Determine the mechanism of cell cycle arrest caused by p21 induction; 2) determine the 4 types of macrophages obstructing the sinusoids that cause centrilobular hypoxic injury, and 3) determine the epigenetic transformation that characterizes balloon cell degeneration of hepatocytes. The approach is innovative primarily because it utilizes new technologies described above, developed in the applicant's laboratory, enabling for the first time, the direct study of liver biopsies from patients with alcoholic hepatiis. The proposed research is significant because it is expected that when the mechanisms involved in alcoholic hepatitis are understood it will be possible to design therapies that are life saving with major cost savings that are currently expended in the treatment of alcoholic hepatitis.