Molecular mechanisms of transdifferentiation

转分化的分子机制

基本信息

批准号：
7630657
负责人：
JACKIE R BICKENBACH
金额：
$ 9万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-10 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7630657
关键词：
Alzheimer&apos s Disease Antibodies Antigens Autologous B-Lymphocytes Bone Marrow Cause of Death Cell Aging Cell Line Cell Lineage Cell Surface Proteins Cell membrane Cell surface Cells Characteristics Coculture Techniques Complement Complement Factor B DNA DNA Sequence Rearrangement Data Detection Diabetes Mellitus Disease Ensure Environment Enzyme Gene Enzymes Erinaceidae Event Exhibits Exposure to Future Genes Green Fluorescent Proteins Hand Heart Diseases IGH@ gene cluster Immunoglobulins Individual Intermediate Filaments Keratin Kinetics Label Ligands Lymphocyte Mediating Membrane Mesenchymal Molecular Monitor Mus Mutation Numbers Organ Personal Satisfaction Phenotype Polymerase Chain Reaction Population Process Proteins Proto-Oncogene Protein c-kit Skin Staging Stem cells Stromal Cells Testing Time Tissues V(D)J Recombination abstracting adult stem cell cell age cell type clinically significant cytokine embryonic stem cell epidermis cell keratin 14, K14 keratin 5 keratinocyte notch protein paired box 5 protein (B-cell lineage specific activator)panacea protein B receptor recombinase response tissue regeneration transcription factor transdifferentiation

项目摘要

ABSTRACT Transdifferentiation has become a common claim for somatic stem cells, yet how this is accomplished has not been well investigated. Although much evidence exists that somatic stem cells can change their protein phenotype, it is not clear what mechanisms are involved, and whether the stem cells actually change their function and maintain this change. To investigate the mechanisms of transdifferentiation, we propose to use two cell types with distinct characteristics and functions: the epidermal stem cell (EpiSC) and the B lymphocyte. EpiSCs express the intermediate filaments keratins 5 and 14 and form sheets of cells connected by adherens and desmosomal junctions. B lymphocytes, in contrast, do not express keratin intermediate filaments. They express a defined set of cell surface proteins with well-studied kinetics of expression. In order for a B lymphocyte to produce a specific immunoglobulin, it must incur a permanent genetic change. This involves deletion and rearrangement of VDJ segments in their immunoglobulin heavy chain locus. This rearrangement is mediated by the RAG1 and RAG2 recombinase enzymes, and by the Pax5-encoded transcription factor BSAP (B cell specific activator protein). EpiSCs do not exhibit VDJ rearrangement; express the RAG1, RAG2, or Pax5 gene; or produce immunoglobulin. Our preliminary data suggest that EpiSCs can be directed to express the B lymphocyte cell markers and genes, and show rearrangement of VDJ segments. We hypothesize that EpiSCs are developmentally flexible, i.e. that they can be directed to produce cells of alternate lineages. To test this hypothesis, in Aim 1, we propose to direct EpiSCs (marked with ¿gal) to transdifferentiate into B lymphocytes. We will verify that individual EpiSCs did transdifferentiate by detection of B Cell surface markers and recombinase enzyme genes, and by PCR analysis for VDJ rearrangement in ¿gal- expressing cells. Since permanency of the transdifferentiation event is important for future therapy, we will test whether or not EpiSCs that alter their cell lineage remain transdifferentiated when removed from the B cell inductive environment. In Aim 2, we will attempt to determine the mechanism(s) by which individual EpiSCs transdifferentiate. Our preliminary findings indicate that EpiSCs require direct contact with the S17 stromal cells in order to transdifferentiate. We hypothesize that S17 cells induce EpiSCs to alter their lineage via receptor- ligand interaction(s). We will test this hypothesis by examining the membranes of EpiSCs, before and during co-culture with S17 cells, for the expression of candidate receptor-ligand pairs. Since the skin is the largest organ with potentially the greatest number of stem cells, understanding the mechanism behind transdifferentiation of these cells is a step toward their use in tissue regeneration.

抽象的跨分化已成为躯体干细胞的普遍主张，但是如何完成这项方尽管有很多证据表明体细胞可以改变其蛋白质表型，尚不清楚涉及哪些机制，以及干细胞是否真的改变了它们功能并维护此更改。为了调查转化的机制，我们建议使用具有不同特征和功能的两种细胞类型：表皮干细胞（EPISC）和B 淋巴细胞。 Episcs表达中间丝角蛋白5和14，以及连接的细胞表由附着和脱染色体连接。相反，B淋巴细胞不表达角蛋白中间体细丝。他们表达了一组定义的细胞表面蛋白，具有精心研究的表达动力学。为了为了使B淋巴细胞产生特定的免疫球蛋白，必须引起永久性的遗传变化。这涉及其免疫球蛋白重链基因座中VDJ段的删除和重排。这重排由RAG1和RAG2重组酶介导，并由PAX5编码转录因子BSAP（B细胞特异性激活蛋白）。 EPISC不表现出VDJ重排；表达 RAG1，RAG2或PAX5基因；或产生免疫球蛋白。我们的初步数据表明，episcs可能是指示表达B淋巴细胞细胞标记和基因，并显示VDJ段的重排。我们假设EPISC是在开发灵活的，即可以指示的替代血统。为了检验这一假设，在AIM 1中，我们建议将Episc（标记为�Gal标记）转分化成B淋巴细胞。我们将验证单个episcs确实通过检测到转变 B细胞表面标记和重组酶基因，并通过PCR分析进行VDJ重排表达细胞。由于转分解事件的永久性对于将来的治疗很重要，我们将测试从B细胞中删除时，是否会改变其细胞谱系的Episc仍会转变归纳环境。在AIM 2中，我们将尝试确定单个Episc的机制转分化。我们的初步发现表明，EPISC需要与S17基质细胞直接接触为了翻译。我们假设S17细胞会影响EPISC，以通过受体来改变其谱系配体相互作用。我们将通过检查Episcs的膜，之前和期间检查该假设与S17细胞共培养，以表达候选受体配体对。由于皮肤是最大的有可能是最大数量干细胞的器官，了解背后的机制这些细胞的转分化是迈向组织再生的一步。