Characterization of follicular stem cells in Tg.AC mice

Tg.AC 小鼠滤泡干细胞的表征

• 批准号: 6432236
• 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/6432236
• 关键词: apoptosis; carcinogen testing; chemical carcinogenesis; cutaneous papilloma; enzyme inhibitors; gene induction /repression; genetically modified animals; hair follicle; hair follicle disorder; laboratory mouse; mutagen testing; mutagens; neoplasm /cancer genetics; oncogenes; phorbols; skin neoplasms; transforming growth factors

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.

研究表明，v-Ha-ras Tg.AC小鼠在特定的化学物质、全身创伤和紫外线照射下会发生皮肤肿瘤，肿瘤的发生依赖于转基因表达的激活。以前的工作已经证明，可以在一些人认为是干细胞再生的增生性毛囊区域检测到转基因表达。这些研究的目的是利用ras转基因在Tg.AC小鼠皮肤中的可诱导表达作为鉴定和表征毛囊中潜在干细胞种群的一种手段。我们已经通过使用通过毛毡轮磨损去除毛囊间表皮来发展转基因表达，这是这些小鼠毛囊起源的鳞状病变的直接证据。此外，我们通过原位杂交发现，早在磨损后6天，转基因就在重新上皮化的皮肤中表达，这强化了潜在的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具有哪些特征，为由造血启动子驱动的转基因的转录激活提供了允许的微环境的问题，我们证明了CD34，一种造血干细胞标记物，在毛囊中表达的区域与假定的干细胞的位置一致，我们已经在包括Tg.AC在内的7个不同品系的小鼠中显示了这种表达。我们使用流式细胞术对C57BL/6，FVB/n和Tg.AC小鼠的CD34+：Alpha-6整合素+细胞群进行了分选。这一群体约占总细胞的6%，这与干细胞群体的预期相似。为了确定这些细胞是否具有干细胞特征，细胞被放置在集落形成试验中，以及在旨在确定总增殖能力的培养中。为了确定CD34+细胞是否在Tg.AC小鼠和使用FVB/N和C57BL/6菌株的多阶段表皮癌变模型中都是化学致癌物的靶点，我们建议结合使用流式细胞术、免疫组织化学、激光捕获显微解剖、序列分析和RT-PCR来确定这些细胞是否是潜在的潜在肿瘤细胞。