| Objective: IR(ischemic retinopathy)is a class of retinal diseases characterized by pathological neovascularization after retinal ischemia.IR includes diabetic retinopathy,retinopathy of prematurity,retinal vein occlusion,and wet macular degeneration.The mechanism of IR is complex and has not been fully elucidated.Our study aims to: 1.To apply cell sources for ischemic retinopathy in vitro experiments by isolating and culturing primary rat retinal microvascular pericytes and endothelial cells.2.To explore the method of making complete and reproducible retinal vascular mount and observe retinal vascular morphology under different conditions.3.To screen out new possible therapeutic targets for diabetic retinopathy through text mining.To identify the target genes of miR-494 through bioinformatic analysis.4.To explore the potential molecular mechanism of miR-494 regulating ischemic retinopathy.Methods: 1.Microvascular fragments were isolated from the retina of 3-week-old SD rats by mechanical shearing,trypsin digestion,and cell filter sieve filtration.Primary pericytes and endothelial cells were purified by culture in different selective media and differential digestion.The morphology of pericytes and endothelial cells was observed by phase-contrast microscopy,and the expression of cell markers was detected by immunofluorescence staining.2.The modified technique,such as toothless tweezers combined with glass rod cleaning the vascular network,was used to prepare trypsin-digested retinal vasculature mount in rats and mice of different ages and feeding conditions.The morphological characteristics of the retinal vascular network stained with PAS were observed,and the morphological characteristics and number of string vessels were observed.3."Diabetic retinopathy" and "diabetic Retinopathy" were retrieved from the pub Med2 Ensemble website to obtain their related genes,and the intersection genes of the two were used for subsequent gene ontology analysis and pathway enrichment analysis.String and Cytoscape tools were used to draw the protein interaction network diagram of the protein encoded by the intersection genes and construct the gene module,and the genes screened from the module were used for subsequent drug analysis.The miRNA-m RNA interaction prediction tool discovered the target genes of miR-494 in 7 miRNA databases.4.Diabetic and oxygen-induced retinopathy models were established using C57BL/6J mice.Primary retinal pericytes were cultured in a high glucose medium in vitro to simulate hyperglycemic injury,and retinal endothelial cells treated with cobalt chloride were used to simulate hypoxia injury.To explore the regulatory effect of miR-494 on ischemic retinopathy: the expression levels of target genes and proteins were detected by q RT-PCR and WB.The pathological characteristics of retinal vessels and the number of string vessels were observed by retinal fluorescence plate,trypsin digestion plate,and HE staining.Results: 1.Retinal microvascular pericytes and endothelial cells migrated from the microvascular fragments after 48 hours of primary culture and reached 80% confluency in 14 to 16 days.The pericyte cell body was large and irregular,with a triangular shape and many long processes.The pericyte cell marker α-SMA was positively expressed,and v WF and GFAP were negatively expressed.Endothelial cells showed a flat polygonal shape,positive expression of v WF,and negative expression of α-SMA and GFAP.2.The improved technique successfully preserved the integrity of the retinal vascular network with a low operating cost and a high success rate.It was found that the number of string vessels in the retinal vessels(vascular development)at 6 days after birth was the highest,and the number of string vessels decreased at6 days,2 weeks,and 3 weeks after birth.There was no significant difference in the number of 4 weeks,6 weeks,and 8 weeks,and the number decreased at 24 weeks.In addition,the retina of the mouse model of oxygen-induced retinopathy showed many microaneurysms,and the number of string vessels was significantly more than that of normal mice of the same age.3.According to the keywords "diabetic retinopathy" and "diabetic mellitus",the related genes were 758 and 1430,respectively,and the intersection genes were 536.Gene ontology and pathway enrichment analysis were used to screen out 291 genes.8 key genes and14 drugs were matched by protein interaction network and module analysis,and drug screening network.PTEN,FOXJ3,and DCAF7 are potential target genes of miR-494.4.In DR and OIR models,miR-494 was up-regulated,while DJ-1 and PTEN expressions were down-regulated;In pericytes cultured with high glucose,miR-494 was up-regulated,while DJ-1 and PTEN expressions were down-regulated.The dual-luciferase reporter indicated that DJ-1 and PTEN were the target genes of miR-494.Knockdown of miR-494 reduced the migration number of retinal endothelial cells,the number of retinal string vessels in diabetic mice,and the retinal neovascularization in oxygen-induced retinopathy mice.Conclusion: 1.The use of mechanical shearing and trypsin digestion combined with a cell strainer,selective medium culture and differential digestion can obtain relatively pure retinal pericytes and endothelial cells,providing a primary cell source close to the performance of living cells for ischemic retinopathy in vitro experiments.2.The modified method can produce complete,clear,and successful retinal vascular slices.String vessels may be involved in angiogenesis during vascular development or pathological conditions.3.Eight key genes of diabetic retinopathy were screened and matched to 14 potential drugs for the treatment of diabetic retinopathy.Potential target genes of miR-494 include PTEN,FOXJ3,and DCAF7.4.Mi R-494 could inhibit the expression of DJ-1 and PTEN genes and aggravate ischemic retinopathy.Targeting miR-494 may be a new therapeutic strategy for ischemic retinopathy. |
PDF Full Text Request