Investigating type 1 diabetes pathogenesis using the live pancreas tissue slice platform

1型糖尿病(T1D)是一种自身免疫性疾病，导致产生胰岛素的β细胞被破坏 胰腺的组织。T1D的发展和进展的时间线是不同的，诊断发生在 一些患者的年龄为几岁，而另一些患者在确诊前已有数年的自身抗体。在……里面 T1D时，发生免疫失调，导致自身抗体产生，免疫细胞渗入胰岛 (胰腺炎)，并最终失去β细胞。在这段时间内在β单元格内发生的事件以及由此产生的 对β细胞功能的影响尚不清楚。这项提案的目标是确定β细胞的功能 在T1D发育过程中，特别是在关键的早期无症状阶段。 了解β细胞或胰岛内的这些功能变化对于全面了解 疾病进展和确定可能的治疗靶点。我的总体目标是 建议使用活的胰腺组织切片来探索β细胞在T1D发育过程中的功能， 具体地说，我将确定免疫细胞渗透在β细胞功能障碍和丢失中所起的作用。活体胰腺 组织切片是这些研究的理想选择，因为这些样本将胰岛保持在其自然微环境中 并保存现有的组织病理学，允许研究β细胞功能和免疫细胞 人口。我假设T1D进展早期的免疫失调会导致β功能障碍 细胞葡萄糖代谢途径主要由线粒体应激介导，导致MHC-I类 高表达并增加β细胞对免疫系统的可见性。为了验证这一假设，活体胰腺 从人类器官捐赠者组织产生的组织切片将被使用，并由由 T1D模型小鼠的胰腺组织。共聚焦显微镜技术将被用来确定 同时评估组织内的胰岛素炎和胰岛功能。其他功能评估 如灌流实验之后的胰岛素ELISA和基因表达将被用来观察 功能更详细。该提案的第一部分侧重于为确定 原位免疫细胞对β细胞功能的影响以获得对疾病时间表的更多控制，特别是 研究疾病的早期阶段，人类白细胞抗原匹配的T细胞替身将被引入来控制人类 胰腺组织切片，创造了一个与世隔绝的环境。疾病的发生和发展将被研究 进一步对NOD小鼠模型的脾细胞进行过继移植实验。进展到T1D 随着疾病的发展，将用活的胰腺组织切片进行监测。功能评估 免疫细胞研究将如上所述进行。我期待着拟议的 研究将确定免疫失调在β细胞功能障碍中所起的作用以及 β细胞功能障碍的机制起源。这将提供有关T1D如何发展的关键信息 并将指明可能的治疗目标，以阻止疾病的进展。

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of the insulin-producing β cells of the pancreas. The timeline of T1D development and progression is heterogenous with diagnosis occuring at a few years of age in some patients versus other patients having autoantibodies for years before diagnosis. In T1D, immune dysregulation occurs leading to autoantibody production, immune cell infiltration into the islets (insulitis), and eventual loss of β cells. The events that occur within the β cell during this time and the resulting impacts on β cell function remain unknown. The goal of this proposal is to determine how β cells function during the course of T1D development, particularly during the critical early, asymptomatic stages. Understanding these functional changes within β cells or islets is critical for a complete understanding of disease progression and for the identification of possible therapeutic targets. The overall objective of my proposal is to use live pancreas tissue slices to explore how β cells function during T1D development, specifically I will identify the role immune cell infiltration plays in β cell dysfunction and loss. Live pancreas tissue slices are ideal for these studies because these samples keep the islet in its native microenvironment and preserve the extant tissue pathologies, allowing for studies of both β cell function and immune cell populations. I hypothesize that immune dysregulation early in T1D progression results in dysfunction of the β cell glucose metabolism pathway primarily mediated by mitochondrial stress, resulting in MHC class I hyperexpression and increased β cell visibility to the immune system. To test this hypothesis, live pancreas tissue slices generated from human organ donor tissue will be used and complemented by slices made from pancreata of mouse models of T1D. Confocal microscopy techniques will be used to determine the degree of insulitis within the tissue while islet function is simultaneously assessed. Additional functional assessments such as perifusion experiments followed by insulin ELISAs and gene expression will be employed to look at function in greater detail. The first part of this proposal focuses on assessments conducted to determine the impacts of in situ immune cells on β cell function. To gain more control over the disease timeline, particularly to investigate the earliest stages of disease, HLA-matched T cell avatars will be introduced to control human pancreas tissue slices, creating an insulitic environment. Disease initiation and progression will be studied further through adoptive transfer experiments with splenocytes from NOD mouse models. Progression to T1D will be monitored with live pancreas tissue slices being made as disease develops. Functionality assessments and immune cell studies will be conducted as discussed above. I expect the contribution of the proposed research will be the identification of the role that immune dysregulation plays in β cell dysfunction as well as the mechanistic origination of β cell dysfunction. This will provide critical information about how T1D develops and will indicate possible therapeutic targets to halt disease progression.