MOLECULAR CHARACTERIZATION--EPIDERMAL GERMINATIVE CELLS

描述(改编自申请人的摘要)：长期目标 这个项目是为了识别上皮细胞表达的基因 这样我们就可以更好地了解它们的生长和 调控及其与癌症发展的关系。使用 抑制性PCR消减杂交法，我们分离到了 在从培养的外周血细胞建立的细胞系中差异表达 来自毛囊区域的根鞘细胞(SV-ORSI) 包含毛囊的凸起区域。一个全长的cDNA 代表这些克隆(称为T27)中的一个的克隆已被获得，并且通过 序列分析，它代表了一个以前没有描述过的基因。T27是 在干细胞丰富的上皮组织中表达，包括睾丸 肠隐窝，包皮组织中深网的尖端和 与头皮组织中毛囊突起一致的区域。它也是 在培养的外根鞘细胞中表达。我们将在现场使用 杂交/原位逆转录聚合酶链式反应和免疫组织化学进一步确定T27 已知卵巢癌上皮组织中mRNA和蛋白水平的表达 干细胞群体和肿瘤组织中。表达式将相互关联 具有克隆形成能力。EM放射自显影/T27免疫金染色法 抗体将被用来检测T27在慢循环中是否表达 包皮和头皮组织中的细胞，并确定其形态。这个 T27蛋白的功能(例如.独特的细胞表面分子，生长 因子及其受体或细胞骨架蛋白)将在 用T27表达载体建立的SV-ORS1细胞和In细胞系 由可诱导的启动者指导。PK活性、细胞内定位 并将研究其与细胞周期的关系。我们将使用反义 抑制T27表达的质粒和寡核苷酸及其检测 对细胞增殖的影响。用来自T27的引物进行聚合酶链式反应 编码序列，我们已经从基因组文库中分离到两个克隆。我们 建议使用这些克隆来确定位置、核苷酸序列 和T27基因的组织。来自我们的另一个的全长克隆 将获得候选基因，并对它们的表达进行评估，类似于 T27.对上皮干细胞所阐述的分子的洞察可能会 对我们理解上皮细胞增殖的广泛影响/ 分化与癌变。它们在基于干细胞的研究中很重要 基因疗法。患有增殖性组织疾病的患者，如 脱发、基底细胞癌和慢性伤口也可能受益于 这些研究。

DESCRIPTION (Adapted from the applicant's abstract): The long term goals of this project are to identify genes that are expressed by epithelial germinative cells so that we may better understand their growth and regulation and their relationship to cancer development. Using a suppression PCR subtractive hybridization method, we isolated clones that are differentially expressed in a cell line established from cultured outer root sheath cells (SV-ORSI) derived from a hair follicle region that encompasses the bulge area of the hair follicles. A full length cDNA representing one of these clones (termed T27) has been obtained, and by sequence analysis, it represents a previously undescribed gene. T27 is expressed in stem cell-enriched epithelial tissues including testis, the intestinal crypt, the tips of the deep rete ridges in foreskin tissue and an area consistent with the hair follicle bulge in scalp tissue. It is also expressed in cultured outer root sheath cells. We will use in situ hybridization/ in situ RT-PCR and immunohistochemistry to further define T27 expression at the mRNA and protein levels in epithelial tissues with known stem cell populations and in tumor tissues. Expression will be correlated with clonogenic potential. EM autoradiography/immunogold staining with T27 antibody will be used to examine whether T27 is expressed in slow cycling cells in foreskin and scalp tissues and determine their morphology. The function of the T27 protein (e.g.. unique cell surface molecule, growth factor and its receptor or cytoskeleton protein ) will be examined in SV-ORS1 cells and in cell line established with a T27 expression vector directed by an inducible promoter. PK activity, intracellular localization and the relationship to cell cycle will be studied. We will use antisense plasmids and oligonucleotides to inhibit T27 expression and determine its effect on cell proliferation. Using PCR with primers derived from the T27 coding sequence, we already isolated two clones from a genomic library. We propose to use these clones to determine the location, nucleotide sequence and organization of the T27 gene. Full length clones from our other candidate genes will be obtained, and their expression evaluated like for T27. Insights into molecules elaborated by epithelial stem cells could have broad implications on our understanding of epithelial proliferation/ differentiation and carcinogenesis. They are important in stem cell based gene therapy. Patients with disorders of proliferating tissues such as alopecia, basal cell carcinoma and chronic wounds may also benefit from these studies.