早期糖尿病视网膜病变中eEF2K活性变化的实验观察

Activity changes of eukaryotic elongation factor-2 kinase in early stages of diabetic retinopathy

目的观察早期糖尿病大鼠视网膜中真核生物延伸因子2激酶(e EF2K)的变化,探讨e EF2K活性变化在早期糖尿病视网膜病变(DR)中的意义。方法建立SD大鼠链脲佐菌素(STZ)糖尿病高血糖模型,造模成功后,设立糖尿病大鼠(DM)组和正常对照(NC)组。qRT-PCR检测DM组和NC组4周和8周时大鼠神经视网膜中e EF2K的表达情况。免疫荧光法观察定位神经视网膜中e EF2K激活标志物-磷酸化真核生物延伸因子2(p-eEF2)和Müller细胞活化标志物胶质纤维酸性蛋白(GFAP)表达情况。结果 4周和8周时DM组大鼠神经视网膜中e EF2K的表达水平与NC组相比无明显变化。8周时DM组大鼠神经视网膜各层均出现e EF2K激活表现,即p-eEF2表达增强。神经节细胞层出现细胞凋亡样改变,凋亡样神经节细胞中e EF2的磷酸化水平增强最为明显。神经视网膜中Müller细胞活化,GFAP蛋白表达增强。结论 STZ诱导糖尿病大鼠早期视网膜病变中神经视网膜内e EF2K激活,在凋亡样神经节细胞中e EF2K活性上升最为显著。

Objective To investigate the changes of eukaryotic elongation factor 2 kinase (eEF2K) in retina of diabetic rat at early stage, and discuss the role of eEF2K at early-stage diabetic retinopathy.Method Diabetic model was induced by streptozotocin in SD rat, and separated into diabetic model (DM) group and normal control (NC) group accordingly to blood glucose levels. qRT-PCR was applied to study the expression of Eef2k in rat neuroretina at 4 weeks and 8 weeks in DM and NC group. Immunofluorescence was adopted to examine the localization and expression of phospho-eukaryotic elongation factor 2 (p-eEF2), an indicator of eEF2K activity, and glial fibrillary acidic protein (GFAP), a marker of Müller cell activation in neuroretina.Results There is no significant change of the expression of Eef2k mRNA in neuroretina of DM group rat at 4 weeks and 8 weeks in comparison with NC group rat. An elevation of the phosphorylation of eEF2 was detected in different layers of neuroretina of DM rat at 8 weeks, indicating the activation of eEF2K. Apoptotic-like changes in cells were observed in ganglion cell layer (GCL), and a robust increase of eEF2 phosphorylation was present in these cells. Moreover, Müller cells were activated in neuroretina, and the expression of GFAP was increased.Conclusion eEF2K was activated in neuroretina of early-stage diabetic rat induced by STZ, and a robust increase of eEF2K activity was detected in apoptotic ganglion cells.