Stromelysin-1在高糖毒性导致的胰岛微血管内皮细胞功能损伤中的作用及机制研究

Diabetes mellitus is one of the most pressing public health threats in China, whose mechanisms have not been fully clarified. Our previous data demonstrated that both type 1- and type 2- diabetic mice revealed pathological phenotype of impaired pancreatic islet microvascular vasomotion. Furthermore, islet microvascular endothelial cells (IMECs) are demonstrated as the elementary and executive units of islet microvascular vasomotion. We provided evidence that the glucose toxicity may be involved in the impairment of functional status of pancreatic islet microvascular vasomotion, partially through resulting IMECs dysfunction (including barrier function, tube formation and migration capacities, permeability), down-regulating the expressions of eNOS and p-eNOSser1177 and the uncoupling of eNOS. Additionally, we investigated the differences in gene expression profiles in glucose toxicity exposed IMECs and found that stromelysin-1 was up-regulated. Considering that stromelysin-1 is involved in the regulation of microvascular endothelial cells function, we speculate that stromelysin-1 may be related with glucose toxicity derived IMECs dysfunction. Based on the mentioned above, CRISPR-Cas9 will be performed to construct stromelysin-1-/- mice and stromelysin-1-/- IMECs respectively in the present study, together with designed experiments to investigate the roles of stromelysin-1 play in glucose toxicity induced IMECs dysfunction and the possible microcirculatory mechanisms hidden behind the pathological processes.

糖尿病是我国重要公共卫生问题之一，发病机制尚未完全阐明。申请人前期在糖尿病小鼠模型中观察到胰岛微血管自律运动功能异常的微循环病理表型；并证实胰岛微血管内皮细胞（IMECs）是胰岛微血管自律运动的执行单元；高糖毒性通过破坏IMECs屏障功能、成管迁移能力及通透性，下调eNOS、p-eNOSser1177表达，促进eNOS二聚体解偶联导致胰岛微血管自律运功功能受损。此外，申请人通过表达谱芯片和生物信息学分析筛选到stromelysin-1在高糖毒性暴露的IMECs中显著上调，因其参与调节微血管内皮细胞功能，故推测stromelysin-1可能参与了高糖毒性对IMECs功能的损伤。本项目拟在前期工作基础上，应用CRISPR-Cas9基因编辑技术建立stromelysin-1-/-的小鼠和IMECs，探讨stromelysin-1在高糖毒性导致的IMECs功能损伤中的作用及其微循环学机制。

糖尿病是我国重大慢性疾病之一。2011-2021年，我国糖尿病患病人数从9000万增长至1.4亿。微循环参与维持生物组织正常生理功能，是具有独特功能的生命单元。近来研究已证实，胰腺整体微循环功能障碍参与了糖尿病发生发展。.课题组开发了基于激光多普勒技术的生物组织微循环功能测评方法和参数体系，并完成了其有效性验证和可视化工程。通过该测评方法，课题组分别证实了1型糖尿病、2型糖尿病小鼠存在胰腺微循环功能异常的病理表型，表现微血流动力学异常（微血流灌注水平、微血管自律运动频率和节律、血流速度降低）和微循环氧水平降低。.胰岛微血管内皮细胞（IMECs）是微血管自律运动的执行单元；高糖毒性通过破坏IMECs屏障功能、成管迁移能力及通透性，下调eNOS、p-eNOSser1177表达，促进eNOS二聚体解偶联导致微血管自律运功功能受损。此外，高糖毒性可损伤IMECs超微结构，IMECs基膜增厚、线粒体肿胀、内外分泌部间距增宽等，导致微血管源胰岛素抵抗。.课题组通过表达谱芯片和生物信息学分析证实stromelysin-1参与高糖毒性对IMECs的损伤，应用CRISPR-Cas9基因编辑技术建立stromelysin-1 KO IMECs，stromelysin-1 KO显着促进了高糖暴露IMECs的增殖、迁移和血管生成，并减弱了单层细胞通透性。此外，stromelysin-1 KO可通过IL-10、瘦素和 TIMP-2 等因子的表达上调和 MMP-2 表达下调发挥IMECs保护作用。.上述基于微循环理论和生物组织微循环功能测评技术的研究工作提供了微循环研究范式，已与国内多家医院和科研单位开展了合作研究范式，取得了较好的转化应用成果。