Lacrimal Gland Regeneration:Identification and Isolation of Progenitor Cells

描述（由申请人提供）：大约一千万美国人，主要是女性，患有干眼病，其由多种原因引起，包括衰老、屈光手术、绝经、自身免疫性疾病和创伤。虽然这些病症的病因各不相同，但所有病症的共同特征是泪腺分泌减少和眼表损伤，导致炎症、刺激和疼痛。目前的治疗方法只是治标不治本，因为它们针对的是眼表，而不是疾病的原因，泪腺分泌减少。大多数患有干眼病的个体可以从泪腺的蛋白质、电解质和水分泌的改善中受益。在干眼症中，泪腺细胞由于凋亡或坏死而减少，因此使用外源性祖细胞或刺激内源性再生来恢复患病泪腺将通过替换丢失的细胞来改善眼表面的健康，并且可以治愈干眼症。我们的实验室是第一个开发出一种可重复的方案，从成年大鼠眶外泪腺祖细胞，可用于修复患病的泪腺分离。这一发现表明，干细胞或能够再生的细胞存在于泪腺中，并且可以被激活以修复受损的组织。泪腺、腺泡、导管和肌上皮细胞的三种分化细胞类型的再生必须通过三种机制之一发生。新形成的分化细胞来源于相同细胞类型的细胞的增殖、来源于不同细胞类型的细胞的增殖或来源于未成熟的、未分化的祖细胞。该项目的总体目标是修复泪腺，恢复其分泌功能，并通过使用成人泪腺祖细胞开发治疗干眼症的新策略。在初步数据中，我们发现少量泪腺细胞的干细胞标志物呈阳性，可以重复分离。我们假设成人泪腺含有少量祖细胞，这些细胞可以在体外或体内诱导分化为功能性泪腺神经，腺泡，导管和肌上皮细胞，可以修复腺体。为了实现这些目标，我们将使用一组干细胞标记物进行免疫组织化学、蛋白质印迹和流式细胞术，以鉴定培养物中的祖细胞，并将这些标记物与其在体内泪腺中的定位进行比较。通过用对泪腺的主要细胞类型具有选择性的抗体进行双标记以确定祖细胞形成所需的细胞类型来确定祖细胞生态位。然后，我们将用溴脱氧尿苷标记来自受损小鼠泪腺的增殖细胞，以跟踪再生过程中的细胞谱系。我们还将使用GFAP和Ascl3转基因小鼠进行谱系追踪。使用一种类似于形成神经球的方案，我们将确定培养的泪腺祖细胞是否形成可用于修复的漂浮球。 公共卫生相关性：数百万美国人患有干眼病。原因是多种多样的，包括老化、屈光手术、绝经、自身免疫性疾病和导致眼表面损伤的创伤，从而导致疼痛、炎症和刺激。干眼没有治愈方法，并且由于泪腺是泪膜的水层的主要贡献者，因此使用干细胞替换患病的泪腺将改善眼表的健康。

DESCRIPTION (provided by applicant): About ten million Americans, predominantly women, suffer from dry eye disease, arising from a variety of causes including aging, refractive surgery, menopause, autoimmune disease, and trauma. Although the etiology of each of these conditions differs, a common feature of all is a decrease in lacrimal gland secretion and damage to the ocular surface resulting in inflammation, irritation, and pain. Current treatments are only palliative as they target the ocular surface, but not the cause of the disease, a decrease in lacrimal gland secretion. Most individuals with dry eye disease could benefit from improved protein, electrolyte, and water secretion from the lacrimal gland. In dry eye lacrimal gland cells are diminished by apoptosis or necrosis, thus restoration of diseased lacrimal gland using exogenous progenitor cells or stimulating endogenous regeneration would improve the health of the ocular surface by replacing lost cells and could cure dry eye. Our laboratory is the first to develop a reproducible protocol to isolate from adult, rat exorbital lacrimal gland progenitor cells that could be used to repair the diseased lacrimal gland. This finding suggests that stem cells or cells capable of regeneration are present in the lacrimal gland and can be activated to repair damaged tissue. The regeneration of the three differentiated cells types of the lacrimal gland, acinar, duct, and myoepithelial cells, must occur via one of three mechanisms. The newly formed differentiated cells are derived from proliferation of cells of the same cell type, derived from proliferation of cells of a different cell type or derived from immature, undifferentiated, progenitor cells. The overall goal of the project is to repair the lacrimal gland, restore its secretory function, and develop a new strategy to cure dry eye by using adult lacrimal gland progenitor cells. In preliminary data we found that a small number of lacrimal gland cells were positive for stem cell markers and could be reproducibly isolated. We hypothesize that the adult lacrimal gland contains a small population of progenitor cells and that these cells can be induced either in vitro or in vivo to differentiate into functioning lacrimal gland nerve, acinar, duct, and myoepithelial cells that can repair the gland. To achieve these goals, we will use a panel of stem cell markers for immunohistochemistry, western blotting, and flow cytometry to identify the progenitor cells in culture and compare these markers with their localization in the in vivo lacrimal gland. The progenitor cell niche will be determined by double labeling with antibodies selective for the major cell types of the lacrimal gland to determine which cell type is needed for progenitor cell formation. We will then label proliferating cells from injured mouse lacrimal glands with bromodeoxyuridine to follow cell lineages during regeneration. We will also use lineage tracing using GFAP and Ascl3 transgenic mice. Using a protocol similar to one that forms neurospheres, we will determine if lacrimal gland progenitor cells in culture form floating spheres that can be used for repair. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from dry eye disease. The causes are varied and include aging, refractive surgery, menopause, autoimmune disease, and trauma causing damage to the ocular surface resulting in pain, inflammation, and irritation. There is no cure for dry eye and as the lacrimal gland is the primary contributor to the aqueous layer of the tear film, replacement of a diseased lacrimal gland using stem cells would improve the health of the ocular surface.