Thioredoxin Inhibitory Protein in Chronic Liver Disease

描述(由申请人提供)：本提案的主要目标是为申请人詹姆斯·P·汉密尔顿医学博士在强大的、支持性的科学环境中提供重要的持续指导，以便他继续发展成为一名完全独立的内科科学家所需的技能。汉密尔顿博士在胃肠病学方面接受了卓越的临床培训，其中包括高级肝病研究奖学金。此外，在迄今为止的职业生涯中，汉密尔顿博士已经将研究兴趣集中在慢性肝病的分子遗传学基础上。为了推进他的学术生涯并建立他的独立性，需要在生物化学、分子生物学、科学研究设计和数据分析方面进行进一步的培训。本研究将探讨硫氧还蛋白抑制蛋白(TXNIP)在慢性肝病发病机制中的表达及其生物学功能。他的假设是，慢性肝炎中TXNIP的激活会导致活性氧物种(ROS)的增加，进而导致肝纤维化。他进一步推测，一旦发生肝硬变，部分由于高甲基化导致的TXNIP表达的丧失，会导致肝细胞的异常生长和增殖。拟议的项目将应用导师的肝纤维化研究的大量专业知识和资源，以及约翰·霍普金斯大学的其他主要合作者的知识和经验。具体地说，当前提案的目标1将在正常、炎症、肝硬变和癌组织以及肝细胞和肝星状细胞系中检测TXNIP在mRNA和蛋白水平的表达。目的2研究血栓素NIP在慢性肝病中的生物学作用。将检测TXNIP诱导的ROS的产生和促纤维化通路的激活。此外，还将确定TXNIP对细胞生长、增殖和凋亡的影响。目的3将研究血栓素NIP甲基化的调节。这一机制将直接评估，使用亚硫酸氢盐测序和甲基化特异性聚合酶链式反应，以及间接使用去甲基化试剂来逆转转录沉默。

DESCRIPTION (provided by applicant): The primary goal of this proposal is to provide the applicant, James P. Hamilton, M.D., with a significant period of continued mentorship in a strong, supportive scientific environment in order for him to continue developing skills needed to become a fully independent physician-scientist. Dr. Hamilton has undergone superior clinical training in Gastroenterology, including a Fellowship in Advanced Hepatology. Moreover, during his career thus far, Dr. Hamilton has already focused his research interest on the molecular genetic basis of chronic liver disease. To advance his academic career as well as to establish his independence, further training is required in biochemistry, molecular biology, scientific study design, and data analysis. The research proposal described herein will investigate the expression and biologic functions of Thioredoxin Inhibitory Protein (TXNIP) in the pathogenesis of chronic liver disease. His hypothesis is that TXNIP activation in chronic hepatitis leads to increased reactive oxygen species (ROS), which in turn lead to liver fibrosis. He further postulates that once cirrhosis has developed, loss of TXNIP expression, due in part to hypermethylation, results in abnormal hepatocyte growth and proliferation. The proposed project will apply the considerable expertise and resources of the mentor's hepatic fibrosis research with the knowledge and experience of other key collaborators at Johns Hopkins. Specifically, Aim 1 of the current proposal will examine TXNIP expression at the mRNA and protein levels in normal, inflamed, cirrhotic and cancerous liver tissues, as well as in hepatocyte and hepatic stellate cell lines. Aim 2 will scrutinize the biological functions of TXNIP in chronic liver disease in vitro. TXNIP-induced generation of ROS and activation of pro-fibrotic pathways will be examined. Furthermore, effects of TXNIP on cell growth, proliferation, and apoptosis will be determined. Aim 3 will investigate the regulation of TXNIP by methylation. This mechanism will be evaluated directly, using bisulfite sequencing and methylation-specific PCR, as well as indirectly, using demethylating agents to reverse transcriptional silencing.