Salivary Gland Development and Regeneration

描述（由申请人提供）：唾液对维持口腔功能和健康至关重要。头颈部癌症患者的放射治疗导致唾液腺细胞损伤，通常是严重的，不可逆转的，并使其萎缩。永久性修复这种损伤是治疗中的下一个医学挑战，可能通过细胞替代来实现。这项建议的长期目标是确定唾液腺中祖细胞的来源，并利用这些细胞恢复受损腺体的唾液腺功能。我们认为，来自涎腺实质导管部分的细胞应包括未固定的祖细胞，因此它们将是细胞移植后再生萎缩唾液腺的一个非常合适的供体细胞群。为了方便鉴定这样的细胞群，我们培育了一种新的转基因小鼠系，该系在唾液腺导管细胞中特异性表达EGFP和Cre重组酶的融合蛋白。EGFP的表达是这些小鼠导管细胞的细胞特异性标记。此外，与来自ROSA26报告系的小鼠杂交后，可以通过LacZ染色来监测导管细胞命运。重组酶对LacZ报告基因的激活在细胞的后代中得以维持，从而追踪细胞谱系。我们的转基因菌株是我们实验计划所依据的独特资源。我们提出了两个具体目标：(1)鉴定唾液腺细胞亚群，其中包括能够重组多种唾液腺细胞类型的祖细胞。使用荧光激活细胞分选将增殖的颌下腺细胞分成两组：egfp阳性的导管细胞和egfp阴性的腺泡细胞。将分析从每个试管池中产生的细胞类型，以确定祖细胞是否确实存在于导管部分。这些实验将验证导管细胞包括适合萎缩唾液腺再生的多能前体细胞的假设，并为其培养建立最佳条件。(2)将培养的前体细胞导入受损的唾液腺，并建立检测这些细胞正确功能的方法。增殖细胞将被引入萎缩小鼠唾液腺。利用免疫组织化学监测细胞存活和增殖情况。为了评估移植细胞对受体器官的功能贡献能力，我们将产生一个表达标记的人唾液蛋白的转基因系，其表达和分泌可以很容易地监测。将注射从该细胞系分离的细胞，并测量其分泌量。确定确定的祖细胞群并证明其在唾液腺中重新填充和功能的能力是确定细胞移植作为萎缩器官潜在治疗效果的关键一步。

DESCRIPTION (provided by applicant): Saliva is critical for the maintenance of oral function and health. Radiation treatment of head and neck cancer patients causes cellular damage in the salivary glands that is usually severe, irreversible, and renders them atrophic. Permanent repair of such damage is the next medical challenge in therapy, and could potentially be accomplished by cell-replacement. The long range goal of this proposal is to identify the source of progenitor cells in the salivary gland and to use these cells for the restoration of salivary function in an impaired gland. We propose that cells from the ductal fraction of the salivary parenchyma should include the uncommitted progenitor cells, and would therefore be a well suited donor cell population to regenerate an atrophic salivary gland after cell transplantation. To facilitate the identification of such a cell population, we have generated a novel transgenic mouse line, which expresses a fusion protein of EGFP and Cre recombinase specifically in the duct cells of the salivary glands. EGFP expression acts as a cell-specific marker of duct cells in these mice. In addition, ductal cell fates can be monitored by staining for LacZ after crossing with mice from the ROSA26 reporter line. Activation of the LacZ reporter gene by recombinase is maintained in a cell's progeny, tracing the cell lineage. Our transgenic strain represents a unique resource on which our experimental plan is based. We propose two specific aims: (1) To identify a subpopulation of salivary gland cells which includes progenitor cells capable of reconstituting multiple salivary cell types. Cells from proliferating submandibular glands will be separated into two populations using fluorescence activated cell sorting: EGFP-positive duct cells, and EGFP-negative acinar cells. The cell types which arise from each pool in vitro will be analyzed to determine whether progenitor cells do reside in the ductal fraction. These experiments will test the hypothesis that duct cells include multipotent precursor cells that are suitable for repopulation of atrophic salivary glands and will establish optimal conditions for their cultivation. (2) Introduce cultured precursor cells into a damaged salivary gland, and establish an assay for the correct functioning of these cells. Proliferating cells will be introduced into atrophic mouse salivary glands. Cell survival and proliferation will be monitored using immunohistochemistry. To evaluate the ability of the transplanted cells to functionally contribute to the recipient organ, we will generate a transgenic line expressing a tagged human salivary protein, the expression and secretion of which can be readily monitored. Cells isolated from this line will be injected and secretion will be measured. The identification of a defined progenitor cell population and demonstration of its ability to repopulate and function in the salivary gland is a critical step in determining the efficacy of cell transplantation as a potential treatment for the atrophic organ.