2型糖尿病患者1-磷酸鞘氨醇代谢异常对肺血管内皮屏障功能的调节作用及机制研究

The impairment of vascular endothelial barrier due to abnormal lipids metabolism induced by diabetes was the pathophysiology foundation of loss of function in diabetic target organs. Pulmonary is an important suffering organ accompany with dysfunction of pulmonary artery endothelial barriers, but the potential mechanism was still unknown. This research aims to figure out the mechanism of dysfunction in diabetic pulmonary in the logic view of diabetes-lipid metabolism-vascular endothelial barrier. In our previous work, we proved that the sphingomyelin metabolism was abnormal in type 2 diabetes mellitus (T2DM) compared with healthy volunteers by the method of 2D LC ToF-MS and the increased level of sphingosine-1-phosphate in T2DM was confirmed by UPLC-MS. In this research, to determine the increasd S1P could regulate the function of pulmonary artery endothelial barrier, we measure the transendothelial electrical resistance by Millicell-ERS Voltohmmeter, the release of intracellular Ca2+ by luminescence spectrometer, the rearrangement of cytoskeletal protein by immunofluorescence microscopy and the phosphorylation of cortical myosin light chain by western blot, considering as the evaluation items of the function of pulmonary artery endothelial barrier; to investigate the specialized S1P receptor involved in the progress above, the function of pulmonary artery endothelial barrier was evaluated after transfection of siRNA targeting S1PR1or S1PR3 separately; to clarify the biological mechanism, the phosphorylation activation of Rac1 or RhoA signal pathway was measured by the methods of pull down and western blot.

糖尿病伴随脂代谢异常造成血管内皮损伤是导致靶器官受损的基础。肺脏作为重要受累靶器官，常出现肺血管内皮屏障破坏，但机制不明。本课题拟从糖尿病-脂代谢-血管屏障角度阐述其机制。前期工作利用2D LC QToF-MS发现2型糖尿病（type 2 diabetes mellitus, T2DM）鞘磷脂代谢异常，利用UPLC-MS证实T2DM中1-磷酸鞘氨醇 (sphingosine 1-phosphate，S1P)含量增高。本课题为评价增高的S1P对肺血管内皮屏障功能的影响，利用电阻仪测定跨内皮细胞电阻，荧光光度计测定胞内钙释放，共聚焦显微镜测定细胞骨架重分布及western blot法测定骨架蛋白磷酸化；利用siRNA对受体封闭，评价肺血管内皮屏障功能后，鉴定参与该过程的S1P受体；为阐述上述生物学机制，通过pull down和western blot实验检测 Rac1及RhoA信号通路的激活。

糖尿病伴随脂代谢异常造成血管内皮损伤是导致靶器官受损的基础。肺脏作为重要受累靶器官，常出现肺血管内皮屏障破坏。本课题拟从糖尿病-脂代谢-血管屏障角度阐述其机制。利用2D LC QToF-MS发现2型糖尿病（type 2 diabetes mellitus, T2DM）鞘磷脂代谢异常，利用UPLC-MS证实T2DM中1-磷酸鞘氨醇 (sphingosine 1-phosphate，S1P)含量增高。通过体外使用评价S1P对肺血管内皮屏障功能的影响，证实S1P对肺血管内皮具有抑制增殖、迁移的功能，即：S1P抑制HBSM细胞的增殖，且呈浓度依赖关系，即：S1P浓度越大，抑制增殖作用越明显。阴性对照组的甲醇（S1P溶剂）对HBSM细胞增殖没有影响。，利用siRNA对受体封闭，评价肺血管内皮屏障功能后，鉴定参与该过程的S1P受体。在该项目的资助下，课题组完成了对鞘氨醇激酶抑制剂功能的评价，并对搭建了高通量测序的组学研究平台。