Notch3调控糖尿病视网膜周细胞凋亡的机制研究

Diabetic Retinopathy (DR) is one of the most serious diseases caused by Diabetes mellitus (DM). Pericyte loss, widely considered as apoptosis, is the hallmark of early stage of DR and could be crucial for initiation of diabetic pathologies. However the mechanism of pericyte apoptosis is still unclear. Notch singaling pathway is involved in the regulation of cell growth, differentiation, apoptosis and survival，while there is no evidence that Notch is involved in the early DR. In our preliminary study, we observed the acellular formation caused by pericyte loss in diabetic model at week 8, together with downregulation of pericyte marker NG2 in retina indicating the pericyte apoptosis. Of note, Notch3 also decreased significantly in retina at the same timepoint. In light of this, Notch3 could possibly be involved in regulating the pericyte apoptosis in DR. To reveal this, we will apply diabetic models (diabetic rat model, pericyte culture under high glucose and pericyte/endothelial cell co-culture under high glucose) to investigate whether Notch3 could mediate the pericyte apoptosis under hyperglycemia regulation. The underlying molecular mechanism of pericyte apoptosis regulated by Notch3 will also be elucidated. In addition, we will further investigate how hyperglycemia regulates Notch3 expression, especially the involvement of Notch3 upstream transcription factor candidate, Elk-1, which could potentially inhibit Notch3 gene transcription. Our study will provide a theoretical basis for the treatment of diabetic retinopathy.

糖尿病视网膜病变是糖尿病最严重的并发症之一。视网膜中周细胞凋亡导致的周细胞丢失是该病变的最早期特征，然而其凋亡机制仍不明确。Notch信号通路参与调控细胞的增殖、分化、凋亡等过程，但在糖尿病视网膜早期病变中的作用并无报道。前期结果表明，在诱导大鼠糖尿病发生后的第8周，其视网膜出现周细胞丢失导致的无细胞血管形成，周细胞标记物NG2的表达随之下降，提示周细胞发生凋亡。与之相关，Notch3的表达水平明显下降，提示Notch3可能参与调控高糖下周细胞的凋亡。在本课题中，我们将分别利用糖尿病大鼠模型、高糖培养周细胞模型以及周细胞内皮细胞共培养模型，首先确定Notch3是否介导高糖引起的视网膜周细胞的凋亡，接着进一步探讨Notch3调控周细胞凋亡的分子机理，最后分析高糖调控Notch3表达的途径并明确转录因子Elk-1对Notch3的负调控作用。通过研究期望为糖尿病视网膜早期病变的治疗提供理论依据。

微血管周细胞的丢失是糖尿病视网膜病变的重要标志和关键原因之一，多数研究认为周细胞的凋亡是丢失的主要途径，然而周细胞的凋亡和丢失发生的时间点往往晚于视网膜的功能损伤，据此我们提出，周细胞作为一类多分化来源，并具备再分化潜能的支持类细胞，其功能的改变对视网膜病变造成的影响可能远远大于或早于丢失造成的影响。因此，本项目拟研究视网膜周细胞功能损伤对糖尿病视网膜病变等视网膜相关疾病的影响以及内在的分子调控机制。糖尿病视网膜病变等多种视网膜相关疾病总伴随低度炎症的发生，尤其是Interleukin-1β的高表达。我们的研究发现在IL-1β模拟的炎症刺激下，视网膜周细胞可发生细胞表型转变，大量表达促炎因子、血管新生因子、迁移相关蛋白等，并下调收缩蛋白、抑制血管新生因子以及与内皮细胞的连接蛋白；功能实验发现IL-1β刺激的周细胞培养上清可趋化小角质细胞，并抑制内皮细胞的紧密连接蛋白和存活等，从而进一步放大视网膜的炎症反应并破坏血-视网膜屏障的稳定性；深入机制研究发现，STAT3信号通路部分介导了视网膜周细胞炎症激活的反应，该过程可被Notch/Hes1信号进一步调控；我们进一步在玻璃体注射IL-1β和缺氧诱导的视网膜血管新生模型中检测到pSTAT3的激活以及大量促炎因子和血管新生因子的表达，提示视网膜周细胞的STAT3信号通路、炎症激活和表型转化在视网膜病变中的可能作用。该研究从周细胞功能转变的角度出发，为相关视网膜疾病的发病机制进行了补充，从而为糖尿病视网膜病变等伴随炎症的视网膜相关疾病的治疗提供了新的切入点和理论依据。