Study On The Gene Expression Profiles And The Pathogenesis Of Corneal Neovascularization

Purpose: Study on differential gene expression profiles in rat CNV, by using microarrays. Hybridization in situ (ISH) and western blot were performed to examine MMP-13, expressed differentially in CNV on the level of mRNA and protein.Methods: l.corneal angiogenesis was provoked by cornea suture. 38 SD rats were subdivided into groups of days 1,4,7,12,24 and 90.Morphologic change and the amount of neovascularization was analysised in sections and corneal flat preparations.2. corneal angiogenesis was provoked by cornea suture. 40 SD rats were subdivided into groups of days 4 and 24.The cornea tissue was processed for Oligo microarrays with 5705 genes and analysised the differentially expressed genes.3. corneal angiogenesis was provoked by cornea suture. 30 SD rats were subdivided into groups of days 1,4, 12, 24 and 90. The cornea tissue was processed for ISH of MMP-13mRNA. 4. corneal angiogenesis was provoked by cornea suture. 18 SD rats were subdivided into groups of days 1, 4, 7, 12 and 24. The cornea tissue was processed for western blot of MMP-13.Results: 1 .slit lamp microscope showed cornea edema on day 1, vessel expension at limbus of cornea; vascular bud appeared on day 4; vessel density and caliber reached to high peak on day 12; vessels regression on day 24 .The amount of CNV in corneal flat preparations: the area of corneal neovascularization increasedgradually within days 1 to 12, the peak phase is on day 12 ( 8.70± 0.76 ) mm~2. 2. there were 57 genes expressed differentially among the 5705 genes more than fourfold on day 4. 29 genes were upregulated versus 28 downregulated genes. The most upregulated genes were SCYA2, S100A9, MMP-3, TIMP-1 and IL1R2 versus the most downregulated genes GALE,GATM and GALD1. There were 38 genes expressed differentially among the 5705 genes more than fourfold on day 12. 34 genes were upregulated versus 4 downregulated genes. The most upregulated genes were MMP-3,MMP-12, MMP-13,TIMP-1,SCYA2 and S100A9 versus the most downregulated genes ALDH1A1.the upregulated genes were significantly more than the downregulated ones 3.MMP-13mRNA was not expressed in normal cornea. It was observed mostly in the corneal stroma cells and inflammation cells on day 4 after suture, the positive staining area for MMP-13mRNA increased from days 4 to 12(p<0.01),and decreased on day 24. 4.The integrated optic density (IOD)of pro-MMP-13 with molecular weight of 60 KDa decreased significantly on days 7 and 12. IOD of active MMP-13 with molecular weight of 48KDa increased gradually from days 1 to 12, and decreased on day 24.Conclusion: cornea suture can be successfully used to provoke CNV experimental model in the SD rat. Genes of MMP-3 , MMP-12, MMP-13, TIMP-K SCYA2, S100A9 involved in the formation of CNV. The expression of MMP-13mRNA situated mainly in cornea stroma cells and inflammation cells after suture stimulating. The similar change on the level of DNA, mRNA and protein demonstrated that MMP-13 may play an important role in...