Characterization Of Follicular Stem Cells In Tg.AC Mice

Tg.AC 小鼠滤泡干细胞的表征

• 批准号: 6534973
• 负责人: Raymond W Tennant
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6534973
• 关键词: CD34 molecule; carcinogen testing; chemical carcinogenesis; cutaneous papilloma; dissection; enzyme inhibitors; flow cytometry; gene induction /repression; genetically modified animals; hair follicle; hair follicle disorder; immunocytochemistry; in situ hybridization; laboratory mouse; neoplasm /cancer genetics; nucleic acid sequence; oncogenes; polymerase chain reaction; skin neoplasms; stem cells

The v-Ha-ras Tg.AC mouse has been shown to develop cutaneous neoplasms in response to specific chemicals, full-thickness wounding, and UV radiation exposure, and tumor development is dependent upon activation of expression of the transgene. Previous work has demonstrated that transgene expression can be detected in a region of hyperplastic hair follicles identified by some as a putative stem cell resevoir. The goal of these studies is to utilize inducible expression of the ras transgene in Tg.AC mouse skin as a means to identify and characterize potential stem cell populations residing in the hair follicle. We have shown through the use of removal of the interfollicular epidermis through felt-wheel abrasion that transgene-expressing develop, which is direct evidence that squamous lesions arise from hair follicle origin in these mice. In addition, we have found transgene expression by in situ hybridization in re- epithelialising skin as early as 6 days following abrasion, strengthening the hypothesis that latent neoplastic cells originate from the hair follicle and express the ras transgene. In order to address the issue of what characteristics follicular stem cells possess that provides a permissive microenvironment for transcriptional activation of a transgene driven by a hematopoietic promoter, we have demonstrated that CD34, a hematopoietic stem cell marker, is expressed in a region of the hair follicle that is consistent with the location of putative stem cells, and we have shown this expression in 7 different strains of mice, including the Tg.AC. Using flow cytometry, we have sorted a CD34+:alpha-6 integrin+ population of cells from C57Bl/6, FVB/n, and Tg.AC mice. This population represents approximately 6% of the total cells, which is similar to what would be expected from a stem cell population. To address whether these cells have stem cell characteristics, cells have been placed in colony forming assays, as well as in culture designed to determine the total proliferative capacity. In order to determine if CD34+ cells are a target for chemical carcinogens both in the Tg.AC mouse and in the multistage epidermal carcinogensis model using FVB/N and C57Bl/6 strains, we propose to use a combination of flow cytometry, immunohistochemistry, laser capture microdissection, sequence analysis, and RT-PCR to determine if these cells are potentially latent neoplastic cells.

已显示v-Ha-ras Tg.AC小鼠响应于特定化学品、全层创伤和UV辐射暴露而发展皮肤肿瘤，并且肿瘤发展依赖于转基因表达的激活。先前的工作已经证明，在增生的毛囊区域可以检测到转基因表达，这些区域被一些人鉴定为假定的干细胞储存区。这些研究的目的是利用Tg.AC小鼠皮肤中ras转基因的诱导表达作为鉴定和表征毛囊中潜在干细胞群体的手段。我们已经表明，通过使用毡轮磨损去除毛囊间表皮，转基因表达的发展，这是鳞状病变产生于毛囊起源在这些小鼠的直接证据。此外，我们已经通过原位杂交发现早在磨损后6天在再上皮化皮肤中的转基因表达，加强了潜伏性肿瘤细胞起源于毛囊并表达ras转基因的假设。为了解决毛囊干细胞所具有的为由造血启动子驱动的转基因的转录激活提供容许微环境的特征的问题，我们已经证明了CD 34，一种造血干细胞标志物，在与推定干细胞的位置一致的毛囊区域中表达，我们已经在7种不同品系的小鼠中展示了这种表达，包括Tg. AC。使用流式细胞术，我们已经分选了来自C57 B1/6、FVB/n和Tg.AC小鼠的CD 34+：α-6整联蛋白+细胞群。该群体约占总细胞的6%，这与干细胞群体的预期相似。为了解决这些细胞是否具有干细胞特征，将细胞置于集落形成测定中，以及置于设计用于确定总增殖能力的培养物中。为了确定CD 34+细胞是否是Tg.AC小鼠和使用FVB/N和C57 Bl/6菌株的多阶段表皮致癌模型中化学致癌物的靶点，我们建议使用流式细胞术、免疫组织化学、激光捕获显微切割、序列分析和RT-PCR的组合来确定这些细胞是否是潜在的潜伏性肿瘤细胞。