CHRARACTERIZATION OF FOLLICULAR STEM CELLS IN TG.AC MICE

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。为了进一步确定Tg.AC小鼠皮肤中乳头状瘤前体细胞的特征，我们从TPA治疗的非肿瘤皮肤中分离出角质形成细胞，并结合使用流式细胞仪和针对毛囊干细胞标志物Beta-one整合素的抗体孵育，结果表明转基因表达与Beta-One阳性细胞是一致的。这进一步证明了卵泡干细胞表达ras转基因。最后，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，在毛囊的一个非常特定的区域表达。由于有证据表明β-酮整合素和CD34在造血干细胞中共表达，我们建议使用流式细胞术、免疫组织化学和激光捕获显微解剖相结合的方法来更密切地定义和表征乳头状瘤前体细胞。-转基因；表皮干细胞；角质形成细胞；激光捕获显微切割

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. To further characterize papilloma precursor cells in Tg.AC mouse skin, we have isolated keratinocytes from TPA-treated, non-tumor bearing skin and using a combination of flow cytometry and incubation with an antibody specific to Beta-one integrin, a putative follicular stem cell marker, have shown that transgene expression is co-incident with beta- one positive cells. This is further evidence that follicular stem cells express the ras transgene. Finally, 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 very specific region of the hair follicle. Since there is evidence that Beta-one integrin and CD34 are co-expressed in hematopoietic stem cells, we propose to use a combination of flow cytometry, immunohistochemistry, and laser capture microdissection to more closely define and characterize papilloma precursor cells. - Transgenic; epidermal stem cells; keratinocyte; laser capture microdissection