表面活性蛋白D介导的Notch信号通路调控急性胰腺炎胰岛β细胞损伤后分化及再生的机制研究

Diabetes mellitus (DM) and impaired glucose tolerance (IGT) after acute pancreatitis (AP) have been attached increasing importance in academia, but its mechanism is unknown. Surfactant protein D (SP-D), an innate immune protein, has been found to be associated with type 2 diabetes, insulin sensitivity as well as tissue regeneration. Under physiological conditions, SP-D is expressed in islets and pancreatic duct but not in acinar cells. Recently, we also observed SP-D expression in SOX9+ cells in the acinar-to-ductal metaplasia (ADM) of acinar injured areas in AP mouse model. In addition, we further observed that a poor blood glucose recovery, rare redifferentiated insulin- β cell, and excessive activated notch signaling occurred in AP SP-D knockout mice compared in AP WT mice. However, it is speculated that injured islet beta-cell differentiation and regeneration in acute pancreatitis could be regulated by surfactant protein D-mediated Notch signaling pathway, and then affect glucose homeostasis. This proposal aims: 1) Establishing the mice AP model, to study the changes of SP-D expression in pancreatic tissues, and the correlation between SP-D and beta-cell differentiation and regeneration. 2) To investigate the effects of SP-D on the redifferentiation of dediferentiated β cells into insulin+ β cells, and the potential conversion of SOX9+ duct-like cells into insulin+ β through regulating Notch signaling pathway by in vitro and in vivo approaches. This project will elucidate the effects of SP-D on injury，repair and regeneration of islet β cells in AP，as well as its molecular/signaling mechanisms, thus providing theoretical basis for the prevention and treatment of AP-induced DM.

急性胰腺炎（AP）后糖耐量受损（IGT）及糖尿病（DM）逐渐引起学界重视，但机制仍不清楚。表面活性蛋白D（SP-D）是一种天然免疫蛋白，被证实与II型DM、胰岛素敏感性及组织再生密切相关。生理情况下，SP-D在胰腺中仅表达于胰岛及胰导管。本团队前期研究发现，AP时胰腺腺泡导管样化生结构SOX9+细胞也表达SP-D。较野生型，SP-D敲基因小鼠AP后Notch通路持续激活，IGT更严重，β细胞去分化后极少再分化。我们推测SP-D可能介导Notch通路调控AP胰岛β细胞损伤后分化及再生而影响血糖稳态。本项目拟开展以下研究：1）建立AP模型，研究SP-D在胰腺的表达及其对β细胞分化及再生的作用。2）通过体内外实验，探讨SP-D调节Notch活性，促使AP后去分化的β细胞再分化及SOX9+细胞转化为功能性β细胞。以期明确SP-D在β细胞损伤后分化及再生中的作用机制，为防治AP后DM提供理论基础。

急性胰腺炎（AP）后糖耐量受损（IGT）及糖尿病（DM）逐渐引起学界重视，但机制仍不清楚。表面活性蛋白D（SP-D）是一种天然免疫蛋白，被证实与II型DM、胰岛素敏感性及组织再生密切相关。生理情况下，SP-D在胰腺中仅表达于胰岛及胰导管。AP时胰腺腺泡导管样化生结构SOX9+细胞也表达SP-D。较野生型，SP-D敲基因小鼠AP后Notch通路持续激活，IGT更严重，β细胞去分化后极少再分化。我们证明了SP-D可介导Notch通路调控AP胰岛β细胞损伤后分化及再生而影响血糖稳态。以下为主要研究内容：1）成功建立AP模型，研究SP-D在胰腺的表达及其对β细胞分化及再生的作用。2）通过体内外实验，验证了SP-D调节Notch活性，促使AP后去分化的β细胞再分化及SOX9+细胞转化为功能性β细胞。结果：1）KO小鼠显示胰腺损伤增加，组织病理学评分更高。2）KO小鼠的IL-1β和IL-6水平在第1天显示更高。急性胰腺炎7天后，KO和WT小鼠之间没有显著差异。3）与CON组相比，我们发现AP7d、AP28d组的AUC显著增加。与WT小鼠相比，KO小鼠表现出更高的空腹血糖水平和更严重的糖耐量受损。特别是在AP后28d。然而，在WT和KO小鼠中，AP1d组和CON组之间没有观察到上述参数的显著差异。4）NGN3表达细胞的百分比在28天降低，但在KO小鼠中仍有少数区域存在强烈染色。然而，在WT小鼠中，与CON组相比，28天时胰岛NGN3的水平没有显著变化。5）AP后28天， WT小鼠胰腺中MafA和胰岛素的表达几乎恢复。然而，在KO小鼠中，我们观察到与CON组相比，在治疗后第28天，MafA+细胞的百分比显著降低。6）AP后，Hes1和RBPJ表达显著增加，尤其是在第1天。与WT小鼠相比，SP-D-/-小鼠AP后，受损腺泡区Hes1和RBPJ的表达增加更为显著。这些结果表明，蛙皮素诱导的急性胰腺炎中，当SP-D缺失时，Notch活性显著增强。7）小鼠原代胰岛细胞试验结果显示，SPD基因敲除使SOX9+导管样细胞减少，不利于之后恢复期的再分化。8）在胰岛素瘤细胞系min6中Notch信号通路配体及其受体Nocth1、Delta-like 1与SPD、胰岛去分化的相关性较高。9）min6过表达SP-D能显著减缓MIN6细胞IL-1β所致的Notch1升高。