氧诱导视网膜病变小鼠模型中的蛋白表达谱分析

Background: Retinal neovascularization (RNV) is one of the major causes of blindness worldwide, yet pathogenic mechanisms of this disease remain unclear, and therapeutic modalities are not satisfactorily effective. Therefore, it is necessary to identify ocular protein markers with significant expression changes during RNV and provide novel therapeutic targets for neovascular retinopathies. Purpose: To study retinal vessel morphological characteristics and protein expression profiling in a mouse model of oxygen-induced retinopathy (OIR). Methods: C57BL/6J mouse pups were randomly divided into normal and OIR group at postnatal day 7 (P7). The normal group was raised under room air for 10 d. The OIR group was exposed to (75±2)% oxygen. The moms were alternated between the two groups every day. The OIR group returned to room air at P12. At P17, mice from both groups were retro-orbitally injected with fluorescein isothiocyanate-dextran (FITC-dextran), and retinal whole mounts were prepared after fixation. The retinal vessels labeled with FITC-dextran were observed and quantified; paraffin sections of eye balls were processed for Hematoxylin and Eosin (H&E) staining, and pre-retinal vascular cell nuclei were quantified. The total proteins were extracted from the eyes, the expression profiling was analyzed by a customized protein array and verified by Western Blot and ELISA. Results: Retinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in OIR group were tortuous, disorganized with neovascular buds, and the avascular area was prominent in the center. Contrastly, the vessels were smooth, organized, and evenly distributed in normal retinas. The percentage of vascular obliteration in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P<0.01, normal vs OIR). The number of pre-retinal vascular cell nuclei, as revealed by H&E staining, was (13.59±4.28) in OIR retina and substantially higher than that (1.48±1.27) in normal retina (P<0.001, normal vs OIR). Protein array showed that 10 out of the 62 examined pro-inflammatory, pro-angiogenic cytokines exhibited more than 1.5-fold expression changes. Among the 10 cytokines, 3 were up-regulated, 7 down-regulated; 4 cytokines showed more than 2-fold expression changes, 3 were down-regulated and 1 was up-regulated. The differential expression was verified by Western Blot and ELISA. The expression trends of platelet factor 4 (PF-4), vascular endothelial growth factor A (VEGF-A), selectin P (SELP), vascular cell adhesion molecule 1 (VCAM-1), soluble tumor necrosis factor receptor II (sTNF-RII) and CXCL16 were consistent with those revealed by protein array. PF-4, VEGF-A, and SELP were up-regulated, and the other 3 down-regulated (P<0.05 normal vs OIR). Conclusion: Differential expression of the cytokines, including PF-4, VEGF-A, SELP, VCAM-1, sTNF-RII and CXCL16 was identified between normal and OIR mouse eyes. These protein expression changes may be associated with RNV. These results would provide new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathies.