【摘要】 目的研究一氧化氮合酶(NOS)在8周糖尿病大鼠视网膜的表达,探讨其与一氧化氮在糖尿病视网膜病变(DR)中可能的分子作用机制。方法采用限制片段差异显示PCR(RFDD-PCR)技术建立正常和8周糖尿病大鼠视网膜基因表达谱,经生物信息学分析两者差异,初步确定NOS三种亚型-eNOS、nNOS和iNOS为DR相关基因,并以半定量RT-PCR和免疫组化方法进行验证。结果RFDD-PCR结果显示,糖尿病组eNOS和nNOS表达下调,iNOS表达上调。RT-PCR结果显示,糖尿病组eNOS和nNOS表达比正常组明显降低(eNOS:0.23±0.03,0.32±0.03,P<0.05;0.25±0.02,0.36±0.02,P<0.05),iNOS比正常组明显增高(0.27±0.02,0.20±0.03,P<0.05)。免疫组化结果显示,正常组eNOS、nNOS和iNOS阳性细胞均见于内核层(INL)和节细胞层(GCL),eNOS阳性细胞也分布于血管内皮层;糖尿病组eNOS和nNOS阳性细胞较正常组明显减少(eNOS:14.33±3.19,22.13±3.60,P<0.05;nNOS:21.87±3.62,34.40±7.09,P<0.05),iNOS较正常组明显增多(17.60±2.58,11.73±2.70,P<0.05)。结论eNOS、nNOS和iNOS表达变化与DR发生发展有关。更多还原

【Abstract】 Objective To investigate the expression of nitric oxide synthase (NOS) in the retina of 8-week-old diabetic rats, and explore the potential molecular mechanisms for the role of NO in diabetic retinopathy (DR). Methods Retinal gene expression profile of normal and 8-week-old diabetic rats was constructed with restriction fragment differential display polymerase chain reaction (RFDD-PCR). Bioinformatic analysis of the differentially expressed gene identified the genes coding for 3 subtypes of NOS, namely eNOS, nNOS and iNOS as the candidate genes related to DR, which was verified using semi-quantitative RT-PCR and immunohistochemistry. Results The results of RFDD-PCR revealed down-regulated expression of eNOS and nNOS and up-regulated iNOS expression in diabetic rat retina. RT-PCR showed that the expression levels of eNOS and nNOS in diabetic rat retina were obviously lower than that in normal retina (0.23±0.03 vs 0.32±0.03 for eNOS, P<0.05; 0.25±0.02 vs 0.36±0.02 for nNOS, P<0.05), but the expression level of iNOS obviously higher (0.27±0.02 vs 0.20±0.03, P<0.05). Immunohistochemistry of healthy retina visualized eNOS-, nNOS-and iNOS-positive cells, all located in the inner nuclear layer (INL) and ganglion cell layer (GCL), and eNOS-positive cells were also found in vascular endothelium. In diabetic retina, the number of eNOS-and nNOS-positive cells was significantly lowered in comparison with normal rat retina (14.33±3.19 vs 22.13±3.60 for eNOS, P<0.05; 21.87±3.62 vs 34.40±7.09 for nNOS, P<0.05), but the number of iNOS-positive cells significantly increased (17.60±2.58 vs 11.73±2.70, P<0.05). Conclusion The alterations in eNOS, nNOS and iNOS expression are associated with the deuelopmant and progression of DR.更多还原