The Role And Mechanism Of CREB On Oxygen Induced Retinal Neovascularization In Mice

Part One Culture and identification of RMVECs and Establishment of a Novel Model of Hypoxia-inducedObjective Primary isolation, culture and identification of retinal microvascular endothelial cells (RMVECs), To establish and evaluate a novel in vitro model of hypoxia-induced retinal nevoscularization.Methods Primary retinal microvascular endothelial cells were isolated from the retinas of C57BL/6J rats and identified by an evaluation for Ⅷ factor related antigen. Cultures were exposed to Bio-bags for0.5,1,2,3,12,24,48and72h. The pO2, pCO2, and pH values of the culture medium in the different hypoxia times were measured with a blood-gas analyzer.Results Ⅷ factor related antigen the identification that cells were>95%pure. When the cells had been exposed to the Biobag for2h, the pO2>2was5.60Kpa, the pCO2was6.04Kpa, and the pH was7.07. When the cells had been exposed to the Biobag for more than2h, the pO2was4.5Kpa, the pCO2and the pH changed slightly.Conclusion Using the tissue blocks adherent method successfully cultured mouse RMVECs.These findings suggested that a novel in vitro model of retinal nevoscularization using the Bio-bag had a good authenticity.. Part Two Effects of CREB on Hypoxia-induced Retinal microvascular endothelial cells apoptosis by adusting PI3K/AKt signal passwayObjective Using RNA interference, To explore the effects of CREB on PI3K/AKt signal passway and to observe the effects of CREB on signal pathway in hypoxia-induced Retinal microvascular endothelial cells apoptosis.Methods Using RNA interference, preparation of CREB targeted siRNA and transfected to RMVECs to detect if the CREB of siRNA can specifically and efficiently knockdown RMVECs CREB gene expression. Observation time is set to the12,24,48,72,96h.The efficacy of the gene transfer was assessed by immunofluorescence staining. The experiment was divided into four groups:normoxia, hypoxia, hypoxia and transfected siRNA group, hypoxia and transfected with empty vector group. Cell proliferation is assay by MTT.RNA and protein expressions of CREB、Akt were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.Results Western blot and PCR showed that the of CREB-siRNA can effectively and specifically knock low RMVECs CREB expression.The most optimal conditions of CREB is the time of72h after the transfection. The transfection efficiency was70%.Real-time PCR shows that hypoxia and CREB-siRNA group expression levels of CREB、Akt mRNA is lower than the other three groups (p<0.05); Hypoxia group and transfected empty vector group is no significant difference (p>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order.Conclusion CREB targeted siRNA in vitro can specifically and effectively inhibit mRNA and protein expression in RMVECs, can be used for follow-up study of gene function. The most optimal conditions of CREB is the time of72h after the transfection. CREB silence can inhibit hypoxia-inducible RMVECs proliferation and induce apoptosis, which may be related to the inhibition of the PI3K/Akt signaling pathway expression. Part Three The expression and significance of CREB in a mouse model of oxygen induced retinopathyPurpose:To establish oxygen induced retinopathy model in C57BL/6J mice by variable oxygen environment and explore the expression and significance of CREB on retinal neovascularization in oxygen induced retinopathy model.Methods:1867-day-old C57BL/6J mice were randomly divided into two groups that normal control group and OIR model group. The normal control group mice were raised in a normal oxygen environment while the OIR model group mice were place into an oxygen-regulated chamber under hyperopic condition(75±2)%O2for5days and were then kept in normoxic condition for a further5days to establish an animal model of oxygen induced retinopathy. OIR model group and normal control group mice were sacrificed at postnatal day17, both eyes were embedded in paraffin and performed HE staining, counting the endothelial cell nucleus which broke through the internal limiting membrane in cross-sections. Both the OIR model and normal control group mice were performed on fluorescein retinal angiography for research of retina vascular morphological changes and retina neovascularization at postnatal day17. Both the OIR mode and normal control group mice were sacrificed at postnatal day7、9、12、14、17and21respectively, Real-Time PCR and Western Blot methods were used to detect the expression of CREB in normal control group retinal and on retinal neovascularization in OIR model group.Results:Compared with the normal control group there were large retinal neovascularization which structure and distribution was disordered and more endothelial cell nucleus which broke through the internal limiting membrane in the OIR model group (P<0.01). The expression of CREB in the OIR model group compared with in the normal control group were significantly decreased in gene and protein levels (p<0.05; p<0.05), and retinal CREB levels were negatively correlated with the progression of retinal neovascularization.Conclusion:The study indicated that the obvious decreased expression of CREB in a mouse model of oxygen induced retinopathy in gene and protein levels, which were high negatively correlated with the progression of retinal neovascularization. CREB may be a direct therapeutic target for ocular neovascularization diseases.