Rac GTPase signaling during embryonic salivary gland branching morphogenesis

描述（由申请人提供）：严重的唾液腺功能减退和口干症（口干）越来越常见，通常是以下原因造成的：1)干燥综合征，一种针对唾液分泌腺泡组织的自身免疫性疾病，2)头颈部癌症的放疗和化疗方案，3)数千种药物的不良副作用。目前基于口干病的治疗是不充分的和暂时的，因此产生了对长期解决方案的重大临床需求，包括用人工工程生物相容性支架上生长的功能性组织替换不可逆转的受损或丢失的唾液组织。然而，在人工工程组织中刺激和/或维持唾液上皮细胞分化的巨大挑战，由于缺乏控制腺体结构和功能的精确分子信号机制的知识而受到阻碍。该研究将通过探索GTPase Rac信号在唾液腺分支形态发生和组织极化的调节中的作用来解决我们对组织形成的理解中的这些空白，这些过程对功能器官的发育至关重要。从这些研究中获得的知识将进一步用于研究Rac在人工工程3D纳米级支架上促进唾液上皮细胞组织和极化的作用，以实现生成功能性人工唾液腺结构的未来目标。具体目的是：(1)确定唾液腺分支形态发生和顶基组织极性的建立是否需要Rac1 GTPase；(2)确定Rac激活是否可以促进人工工程的生物相容性PLGA纳米纤维支架上唾液腺上皮细胞的极化。我们将使用离体全器官培养系统来检测小鼠胚胎颌下唾液腺中Rac1的功能，并使用实时延时或固定共聚焦显微镜来成像分支形态发生和顶基极性的动态。数据还将使用生化免疫印迹和QRT-PCR技术进行分析，并使用成像软件、图像分割和计算细胞图方法进行严格量化。唾液腺疾病，如干燥综合征、唾液腺癌和口干症，都以唾液腺功能减退为病因，这给患者、他们的家庭和整个医疗保健系统带来了巨大的负担。从该项目中获得的唾液腺器官发生早期信号机制的知识将对组织工程和再生医学领域以及未来研究唾液腺疾病中Rac信号的功能和可能的失调具有相当重要的意义。提案中使用的缩写：2D、二维；3 d三维;BM，基底膜；ECM，细胞外基质；鸟嘌呤核苷酸交换因子；GTPase，鸟苷三磷酸羟化酶；IB,免疫印迹;如果,免疫荧光;IP,免疫沉淀反应;Par，分裂缺陷蛋白；聚乳酸-羟基乙酸共聚物；Rac1， ras相关C3肉毒杆菌底物1；SMG，下颌下唾液腺；Tiam1， t细胞淋巴瘤侵袭转移诱导蛋白1。

DESCRIPTION (provided by applicant): Profound salivary gland hypofunction and xerostomia (dry mouth) are increasingly common occurrences and are often the consequences of: 1) Sjogren's Syndrome, an autoimmune disease that targets saliva-secreting acinar tissue, 2) radiation and chemotherapeutic regimens for head and neck cancers and 3) adverse side effects from thousands of medications. Current xerostomia-based treatments are inadequate and temporary thus creating a significant clinical need for long-term solutions that include replacing irreversibly damaged or lost salivary tissue, with functional tissue grown on artificially engineered biocompatible scaffolds. However, the considerable challenge of stimulating and/or maintaining salivary epithelial cell differentiation in artificially engineered tissues has been hindered by the lack of knowledge of precise molecular signaling mechanisms that control glandular structure and function. The proposed study will address these gaps in our understanding of tissue formation by exploring the role of GTPase Rac signaling in the regulation of salivary gland branching morphogenesis and tissue polarization, processes that are crucial for development of a functional organ. The knowledge gained from these studies will further be used to investigate the role of Rac in promoting salivary epithelial cell organization and polarization on artificially engineered 3D nanoscale scaffolds, towards the future goal of generating functional artificial salivary gland constructs. The specific aims are to: (1) determine whether Rac1 GTPase is required for salivary gland branching morphogenesis and the establishment of apico-basal tissue polarity, and (2) determine whether Rac activation can promote salivary epithelial cell polarization on artificially engineered, biocompatible PLGA nanofibrous scaffolds. We will use an ex vivo whole organ culture system to examine Rac1 function in the mouse embryonic submandibular salivary gland and live time-lapse or fixed confocal microscopy to image the dynamics of branching morphogenesis and apico-basal polarity. Data will also be analyzed using biochemical immunoblotting and QRT-PCR techniques and rigorously quantified using imaging software, image segmentation and computational Cell graph methods. Salivary gland diseases like Sjvgren's syndrome, salivary adenocarcinomas and xerostomia, all feature salivary gland hypofunction as a cause, which poses an enormous burden to affected individuals, their families and the health care system as a whole. The knowledge gained from this project on the signaling mechanisms underlying early salivary gland organogenesis will be of considerable significance to the fields of tissue engineering and regenerative medicine and to future studies examining the function and possible deregulation of Rac signaling in salivary gland diseases. Abbreviations used in proposal: 2D, two-dimensional; 3D, three-dimensional; BM, basement membrane; ECM, extracellular matrix; GEF, guanine nucleotide exchange factor; GTPase, guanosine triphosphate hydroxylase; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; Par, partitioning-defective proteins; PLGA, polylactic-co-glycolic acid polymer; Rac1, Ras-related C3 botulinum substrate 1; SMG, submandibular salivary gland; Tiam1, T-cell lymphoma invasion and metastasis-inducing protein 1. PUBLIC HEALTH RELEVANCE: Profound salivary gland hypofunction is a common feature in a majority of patients treated for salivary gland diseases such as Sjvgren's syndrome (SS), salivary neoplasms, adults being treated for head and neck cancer and those taking medications with anti-sialogogue sequelae. Current xerostomia-based treatments are inadequate, temporary and include pharmacological and gustatory stimulants. More long term solutions include tissue replacement and regeneration therapies, however, the significant challenge of maintaining and stimulating epithelial cell organization and differentiation in engineered tissues, remains. Since epithelial cell secretory function is crucial to organ function, understanding the cellular mechanisms regulating and maintaining tissue structure and differentiation is critical to regenerating or engineering functional tissues. The data obtained from this grant will advance basic scientific knowledge regarding novel roles for the small GTPase Rac in the control of salivary gland branching morphogenesis. Utilizing three-dimensional ex vivo whole organ culture systems, we will examine how Rac GTPase-mediated signaling pathways can control major developmental processes such as salivary gland branching morphogenesis and the formation of tissue polarity, an indispensable requirement for unidirectional and controlled flow of saliva. More significantly, the role of Rac in promoting salivary epithelial cell organization and polarization on biocompatible 3D nanofibrous scaffolds will be examined, towards the future goal of creating an artificial salivary gland construct on biocompatible scaffolds for use in regenerative medicine.