高糖诱导的内质网应激在Müller细胞损伤中的作用及其信号转导机制

Müller cell plays an important role in the development of diabetic retinopathy (DR). Endoplasmic reticulum (ER) stress involved in the pathological process of DR. However, little is known about the specific contribution of the Endoplasmic reticulum (ER) stress in Müller cell to the pathogenesis of DR. The purpose of this study is to examine the role of ER stress and the signaling pathway of ER stress-induced by hyperglycemia in Müller cell. Our recent studies have shown that high glucose induced increase of ER stress, ATF4 expression in cultured Müller cells. Based on this finding, this proposal is to define the role of ER stress and the signaling pathway of ER stress-induced by hyperglycemia in Müller cell in vitro (rMC-1), and in vivo (mice with STZ-induced diabetes). The anticipative results will elucidate the signaling pathway of ER stress involved in the pathogenesis of Müller cell and may provide therapeutic potential of ER stress related signaling inhibitors for treatment of DR.

Müller细胞在糖尿病视网膜病变发生发展中起重要作用，内质网应激介导了其病理过程，但具体机制不明。我们的前期研究结果提示，高糖诱导内质网应激反应中重要的信号分子ATF4表达的明显增高。因此，本课题将在此基础上，体外培养Müller细胞株（rMC-1），采用信号分子的化学阻断剂或构建pEGFP-H1/siRNA及pEGFP真核表达载体分别阻断或过表达ATF6，PERK-eIF2α及IRE1，探讨内质网应激在高糖诱导的Müller细胞损伤中的作用及其信号转导途径。此外，应用STZ法建立糖尿病大鼠模型，采用组织学、免疫组织化学及分子生物学等技术手段动物体内实验进一步探讨高糖处理对Müller细胞的损伤作用及其对ERS相关信号分子表达的影响。本课题的最终目的是阐明内质网应激三条信号通道在高糖诱导的Müller细胞损伤中的作用及其信号转导机制，为阐明糖尿病视网膜病变发病机制提供新的理论依据。

本研究主要采用MTT、WESTERN-BLOT、RT-PCR检测rMC-1视网膜Müller细胞在高糖培养环境下，内质网应激的信号分子及细胞的成活率。并构建pEGFP-H1/siRNA及pEGFP真核表达载体分别阻断或过表达ATF6和PERK-eIF2α，检测高糖诱导Müller细胞内质网应激各通道的信号分子。根据本研究的试验结果，我们发现：1.高糖体外培养rMC-1视网膜Müller细胞36h时，细胞抑制率最高；内质网应激通道的信号分子表达量尚未急剧增加，而相关基因的转录却明显增加；2.体外高糖培养视网膜Müller细胞36h，内质网应激信号分子Eif2ak3, ATF4, ATF6, CHOP和GRP78表达量明显升高，Eif2ak3, ATF4, ATF6 和CHOP基因转录量升高；3.我们成功构建ATF6-pEGFP-N1, PERK- pEGFP-N1过表达质粒和ATF6-shRNA, PERK-shRNA抑制表达质粒；