HEPATIC PROGENITORS IN TRANSPLANTATION

描述（由申请人提供）： 候选人：这个职业发展计划旨在完成我作为移植外科医生和免疫和细胞生物学独立研究者的学术生涯的正式准备。该培训计划有三个组成部分：正式的课程，其他教学法，并加强指导，以完成拟议的研究计划的目标。在1-2年：两个免疫学和两个细胞生物学高级课程将采取。这些将在第一年通过密集的研讨会来补充，这将扩大我在分子生物学方面的基础科学技术。每周实验室会议和期刊俱乐部活动将持续进行。在过去的两年里，我专注于学习分离干细胞的原理，这一前期工作是研究计划的一部分。我将与两位导师密切合作，对分离肝干细胞所需的关键实验有更广泛的了解。这个结构化的程序将准备我批判性地思考，提出问题，形成假设，并随后在移植和细胞生物学领域进行创新实验。最后，我作为移植外科医生的临床培训是对我科学追求的理想补充，因为我对开发解决相关临床问题的翻译项目有深刻的见解。 工作环境：北卡罗来纳州大学在校园内拥有多个核心实验室设施，可以完成这些和未来的实验。它们包括荧光激活细胞分选设施，胃肠道和胆道疾病生物学中心，包括先进的细胞技术和组织工程核心，免疫测定核心和共聚焦设施。该提案得到了外科部门的支持，并允许PI将75%的时间用于该项目。大部分工作将在PI的实验室进行。 调研：随着器官的持续短缺，挑战器官移植和缺乏成功的新鲜分离的成熟肝细胞在肝细胞治疗，我提出了一系列的实验的基础上肝祖细胞（与他们的增殖能力），作为一个有前途的替代。为了验证这一假设，我将确定肝祖细胞在移植后的扩张和功能。研究将集中在它们的免疫原性，并将为未来的实验提供基础。 具体目的是：1）鉴定肝祖细胞在细胞扩增过程中肝富集转录因子和肝细胞谱系基因的表达序列; 2）使用绿色荧光蛋白确定肝祖细胞的植入和迁移模式以及随后的细胞分化（转基因）“绿色”小鼠; 3）使用选定的转基因小鼠模型（即鞘磷脂酶缺乏，高胆固醇血症），我将分析肝祖细胞的功能沿着它们在损伤后重建肝脏的能力; 4）分析CD 4+、CD 8+和非T细胞启动同种异体肝祖细胞急性排斥反应的能力。

DESCRIPTION (provided by applicant): Candidate: This career development proposal is designed to complete formal preparation for my academic career as a transplant surgeon and independent investigator in immuno- and cellular biology. The training program has three components: formal coursework, other didactics, and an intensive mentorship to complete the proposed research plan's aims. During years 1-2: two advanced courses in immunology and two in cellular biology will be taken. These will be complemented by an intensive workshop during year 1 that will expand my basic science techniques in molecular biology. Weekly laboratory meetings and journal club activities will be ongoing. During the past two years I have focused on learning the principles of isolating stem cells and this preliminary work is part of the research plan. I will be working closely with both mentors to develop a broader understanding of the critical experiments required to isolate hepatic stem cells. This structured program will prepare me to think critically, ask questions, form hypotheses and subsequently perform innovative experiments in the transplant and cell biology field. Lastly, my clinical training as a transplant surgeon is an ideal complement to my scientific pursuits, as I have insight into developing translational projects that address pertinent clinical questions. Environment: The University of North Carolina has multiple core laboratory facilities on campus that will allow the completion of these and future experiments. They include the Fluorescence Activated Cell Sorting facility, the Center for Gastrointestinal and Biliary Disease Biology that includes the Advanced Cell Technologies and Tissue Engineering Core, the Immunoassay Core, and the Confocal Facility. This proposal is supported by the Department of Surgery and allows the PI to dedicate 75% of his time on this project. The majority of the work will be carried out in the PI's lab. Research: With the ongoing shortage of organs that challenges organ transplantation and the lack of success with freshly isolated mature hepatocytes in liver cell therapy I have proposed a series of experiments based on hepatic progenitors (with their ability to proliferate), as a promising alternative. To test this hypothesis I will determine the hepatic progenitor's ability to expand and function after transplantation. Research will focus on their immunogenicity and will provide a foundation for future experiments. The specific aims are to 1) To identify the sequence of expression of liver-enriched transcription factors and hepatocyte lineage genes in hepatic progenitors during cellular expansion; 2) To determine the engraftment and migration pattern of hepatic progenitors and subsequent cellular differentiation using a green fluorescent protein (transgenic) "green" mouse; 3) Using selected transgenic mouse models (i.e. sphingomyelinase deficient, hypercholesterolemia) I will analyze the function of hepatic progenitor cells along with their ability to reconstitute a liver after injury; 4) Analyze the capacity of CD4+, CD8+ and non-T cells to initiate acute rejection of allogeneic hepatic progenitor cells.