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会导致唾液腺中的细胞损伤，这种损伤通常是严重的、不可逆的，并使它们萎缩。永久修复这种损伤是治疗中的下一个医学挑战，并且可能通过细胞置换来完成。该建议的长期目标是鉴定唾液腺中祖细胞的来源，并使用这些细胞恢复受损腺体中的唾液腺功能。我们建议，从导管部分的唾液实质的细胞应包括未定型的祖细胞，因此将是一个非常适合的供体细胞群再生萎缩的唾液腺细胞移植后。为了便于识别这样的细胞群，我们已经产生了一种新的转基因小鼠系，它表达的EGFP和Cre重组酶的融合蛋白，特别是在导管细胞的唾液腺。在这些小鼠中，EGFP表达作为导管细胞的细胞特异性标记。此外，在与来自ROSA 26报告细胞系的小鼠杂交后，可以通过LacZ染色来监测导管细胞命运。重组酶对LacZ报告基因的激活维持在细胞的后代中，从而追踪细胞谱系。我们的转基因菌株代表了我们实验计划所基于的独特资源。我们提出两个具体目标：（1）鉴定唾液腺细胞的亚群，其包括能够重建多种唾液腺细胞类型的祖细胞。使用荧光激活细胞分选将来自增殖的下颌下腺的细胞分离成两个群体：EGFP阳性导管细胞和EGFP阴性腺泡细胞。将分析来自体外每个池的细胞类型，以确定祖细胞是否确实存在于导管部分中。这些实验将检验导管细胞包括适于萎缩唾液腺再增殖的多能前体细胞的假设，并将建立其培养的最佳条件。(2)将培养的前体细胞引入受损的唾液腺，并建立这些细胞正确功能的测定。将增殖细胞引入萎缩的小鼠唾液腺中。将使用免疫组织化学监测细胞存活和增殖。为了评价移植细胞对受体器官功能性贡献的能力，我们将产生表达标记的人唾液蛋白的转基因系，其表达和分泌可以容易地监测。将注射从该细胞系分离的细胞并测量分泌。确定的祖细胞群的鉴定和证明其在唾液腺中重新增殖和发挥功能的能力是确定细胞移植作为萎缩器官的潜在治疗的功效的关键步骤。

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.