Globle Gene Expression Profile, Cellular And Molecular Mechanism Of Oxygen Induced Retinopathy In A Mice Model

Purpose: Retinopathy of prematurity (ROP) is an ischemia-induced proliferativeretinopathy, which affects premature infants with low birth weight. It is a leadingcause of visual impairment and blindness in children, and shares pathophysiologicalcharacteristics with other common ocular diseases such as diabetic retinopathy,central vein occlusion, and age-related macular degeneration. Much attention has beenpaid on the prevention and treatment of this preventable disorder in the last few years.Among the many features of ROP, neovascularization in nervous system, sufferedin preterm babies, and receiving hyperxia treatment take the 3 most unique ones. TheROP has been explained as the hypoxia induced neovascularization following highdose oxygen delivery. Despite the much information got from an mouse model ofoxygen induced retinopathy(OIR), the detailed mechanism is still leading the interestsof researchers. In this study, we study the development of mouse vasculature, and thegene expression profile in the oxygen induced OIR model, with the intention to findgenes account for the neovascularization. To investigate the regulatory cascade of E2and NGF in retinal vascular endothelial cell, we evaluate the effects of the two,respectively or combined with K252a, one of the Trk A inhibitor,s on RF/6A retinalendothelial cell line. Also on the OIR model, we study the effects of bone marrowstromal cells(BMSCs), by intravitreal transplantation before oxygen exposure. Wealso knock down the BMSC expression of VEGF with a RNA interference plasmid,pSuper-EGFP to study the possible involvement of growth factors, such as VEGF, inROP.Methods: 1. Retina whole mounts of 8 developmental stages selected from postnatal day zero (P0) to P18 mouse were stained with ADPase histo-chemistrical method. Retina serial sections of the same day were used to assess the developmental growth of the retina vasculature. 2. Oxygen induced mouse model of ROP was developed in C57BL mice by a 5-day exposure to 75% oxygen from P 7 trough P12. Pooled total RNA was preparieded with a Trizol method from retinas of the follow groups respectively, P17, P21 and P27 ROP mice together with age corresponded control. Purified mRNA was reverse transcripted to cDNA, and abeled with 6胡宝洋博士学位论文 OIR 基因表达谱及机制 摘要 Cy3 and Cy5 conjuncted neucletide by PCR. Each group of the labeled cDNA was hybridized with an expression profile microarray of oligonucleotides of 16,000 mouse genes. Signal were extracted from the scaned images of chips, followed by background extraction and normalization, and subjected for further analysis. Genes altered 2 fold with statistically significance, from either of the the groups were included in our Gene tree, Condition and SOM cluster anlysis. 3. The estrogen-NGF regulatory cascade was studied on RF/6A retinal vascular endothelial cell line. MTT assay, wound healing assay and ECM matrix gel based tubogenesis assay were carried to study the proliferation, migration and angiogenesis of RF/6A following the administration of estrodial, NGF and inhibitor of Trk A receptor. 4. Bone marrow stromal cells (BMSCs) were separated from the whole marrow cells according to its much more potent adhenrence to the plastic ware. Immunoohistology were used to examine the expression of growth factors, such as VEGF,IGF,bFGF and NGF. The conditioned medium of BMSC, which was enrichment in growth factors, was added to the RF/6A culture medium. Effects on RF/6A were evaluated via the MTT, wound healing and bubogenesis assay after a 24h-incubation. 5. BMSCs of passage 3 to 5 were labeled with CM-DiI 12 hours before transplantation. 1×105 CM-DiI labeled BMSCs in 0.5 ul PBS were intravitreal injected into P7 mi...