Role of Ceramide in Hepatic Stellate Cell Activation and Liver Fibrosis

项目概要/摘要 这是Jennifer Y博士的K 08奖申请书陈，胃肠病学家和肝病学家， 马萨诸塞州总医院。陈博士的长期职业目标是成为一名独立的 资助的医生科学家，投入超过75%的时间来建立和维护一个基础科学 肝纤维化的研究计划。此次K 08奖将为陈博士提供必要的支持， 实现她的短期目标：1）发展分子生物学方面的额外培训，包括研究蛋白质 相互作用和诱导稳定的基因表达; 2）获得全基因组转录分析的专业知识; 3)熟练掌握转基因小鼠工作和肝纤维化小鼠模型; 4）产生数据 及发表纪录。在之前的工作中，候选人已经开发了 一种小分子筛选，以鉴定抑制肝星状细胞（HSC）的化合物， 导致肝纤维化。通过分析一个命中，她确定，鞘脂神经酰胺可以 显著抑制HSC的活化效应子表型。她的研究发现了一种潜在的抗纤维化作用 用于抑制酸性神经酰胺酶（aCD酶），该酶负责神经酰胺代谢。在这一提议中， 候选人试图阐明神经酰胺积累使HSC失活的机制， aCD酶在纤维化进展中的作用。该研究的具体目标是：1）确定神经酰胺 介导HSC失活;和2）确定α CD酶消耗和抑制对纤维化的影响 体内发育根据第一个目标，申请人将利用一系列技术，包括 免疫印迹，小分子抑制和激活，RNA干扰，和组成型表达 激活突变蛋白以了解神经酰胺如何使HSC失活。全基因组表达分析 将进行阐明神经酰胺的转录靶点。根据第二个目标，申请人将 产生具有条件性成纤维细胞特异性aCD酶缺失的小鼠，并将确定 它们在两种独立的肝纤维化模型中经历减少的纤维化。她还将决定 a CD酶的小分子抑制剂在体内的抗纤维化作用。作为这项建议的一个组成部分， 候选人的职业发展将通过参与高级课程和研究来补充 研讨会，以发展分子生物学，全基因组转录分析和体内模型的专业知识 肝纤维化一个正式的指导委员会和咨询团队将提供监督，指导， 帮助候选人实现目标。研究环境，其中包括MGH GI单位， 哈佛纤维化网络、哈佛学院、哈佛医学院和哈佛公共卫生学院， 将提供一个丰富的，合作的，和支持的气氛，以确保候选人的成功。通过这个 获奖后，候选人将成为一名独立的基础科学调查员，为 了解鞘脂神经酰胺的作用及其对离体和体内HSC的失活。

PROJECT SUMMARY/ABSTRACT This is an application for a K08 award for Dr. Jennifer Y. Chen, a gastroenterologist and hepatologist at the Massachusetts General Hospital (MGH). Dr. Chen’s long-term career goal is to become an independently funded physician scientist, devoting more than 75% of her time to establish and maintain a basic science research program in hepatic fibrosis. This K08 award will provide Dr. Chen with the support necessary to achieve her short-term goals: 1) develop additional training in molecular biology, including investigating protein interactions and induction of stable gene expression; 2) gain expertise in genome-wide transcriptional analysis; 3) become proficient in transgenic mouse work and mouse models of hepatic fibrosis; and 4) produce the data and publication record necessary for a successful R01 application. In prior work, the candidate has developed a small molecule screen to identify compounds that inactivate hepatic stellate cells (HSCs), the primary cell type responsible for hepatic fibrosis. Through analysis of a hit, she identified that the sphingolipid ceramide can profoundly inhibit the activated effector phenotype of HSCs. Her work has identified a potential antifibrotic role for inhibition of acid ceramidase (aCDase), the enzyme responsible for ceramide metabolism. In this proposal, the candidate seeks to elucidate the mechanism by which ceramide accumulation inactivates HSCs and define the role of aCDase in fibrosis progression. The specific goals of the study are to: 1) determine how ceramide mediates HSC inactivation; and 2) define the impact of aCDase depletion and inhibition on fibrosis development in vivo. Under the first aim, the applicant will utilize a combination of techniques including immunoblotting, small molecule inhibition and activation, RNA interference, and expression of constitutively activated mutant proteins to understand how ceramide inactivates HSCs. Genome-wide expression analysis will be performed to elucidate transcriptional targets of ceramide. Under the second aim, the applicant will generate mice with a conditional fibroblast-specific deletion of aCDase, and will determine the extent to which they experience reduced fibrosis in two independent models of hepatic fibrosis. She will also determine the antifibrotic effects of a small molecule inhibitor of aCDase in vivo. As an integral part of this proposal, the candidate’s career development will be complemented by participation in advanced coursework and research seminars to develop expertise in molecular biology, genome-wide transcriptional analysis, and in vivo models of hepatic fibrosis. A formal mentorship committee and advisory team will provide supervision, guidance, and assistance for the candidate to achieve her goals. The research environment, which includes the MGH GI Unit, Harvard Fibrosis Network, Harvard College, Harvard Medical School, and the Harvard School of Public Health, will provide a rich, collaborative, and supportive atmosphere to ensure the candidate’s success. Through this award, the candidate will become an independent basic science investigator by contributing to the understanding of the role of the sphingolipid ceramide and its inactivation of HSCs ex vivo and in vivo.