Research On Retinal Neuropathy Of Diabetic Rat

Objective1. Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat with restriction fragment differential display—Polymerase Chained Reaction(RFDD-PCR), and identifying candidate genes related with diabetic retinopathy by bioinformatic analysis of profiles.2. Investigating the levels of mRNA and protein of the candidate factors in the retina of diabetic rats, and imploring the expression changes of candidate factors in the retinal vascular endothelial cells and neurons, and then to provide evidence for clarifying the pathological mechanism of diabetic retinopathy.Methods1. The gene expression profiles of non-diabetic and 8-week Alloxan induced diabetic rat's retina were constructed by RFDD-PCR technique.2. Then all fragments of the two gene expression profiles have been separated and displayed by electrophoresis on 7% urea denaturing polyacrylamide gel and scanned with Typhoon9200 Image system. Image analysis of the gels were carried out by using the software of ImageQuant TL, Image Tool, Fragment Analysis. Candidate genes were selected.3. The expression levels of selected candidate genes were identified by semi- quantitative RT-PCR, Western Blot, immuhistochemistry and Real-time RT-PCR in 8-week diabetic rats.Results1. Having constructed the retinal gene expression profiles of non-diabetic rat and diabetic rat successfully, obtaining 3639 significant fragments, including DR:1886, NR:1813, 840 differential fragment Compared with normal condition, lower signal fragments were 283, higher signal fragments were 225, 145 fragments disappeared, and 187 fragments appeared in diabetic condition.2. The results of Bioinformatic analysis show: compared with normal retina, eNOS and nNOS down-regulated, iNOS up-regulated, ET-1, ET-3, ETRB and ECE up-regulated, ET-2 and ETRA undetected,α-synuclein up-regulated,β- synuclein andγ-synuclein unchanged.3. The results of Semi-quantitative RT-PCR show: compared with normal retina, eNOS and nNOS expressed lower, iNOS, ET-1,ET-2, ET-3, ETRB, ETRB and ECE expressed all higher.4. The results of real-time PCR show: compared with normal retina,α-synuclein,β- synuclein andγ-synuclein expressed all higher.5. The results of Western Blot show: compared with normal retina, the protein levels 3-NT, iNOS, ET, ETRA, ETRB andα-synuclein are all higher, and the protein levels of eNOS, nNOS are lower.6. The results of immuhistochemistry show: compared with normal retina, more 3-NT-positive cells and iNOS-positive cells appear in inner nucleus layer of diabetic retina; less eNOS-positive cells appear in inner nucleus layer and vascular endoderm of diabetic retina; less nNOS -positive cells appear in inner nucleus layer of diabetic retina; more ET-positive cells, ETRA- positive cells, ETRB- positive cells appear in inner nucleus layer of diabetic retina; moreα- synuclein- positive cells appear in ocular cone and rod layer of diabetic retina.Conclusions1. RFDD-PCR is an efficient technique for research diseases genomics as a mass screening to complete gene expression with the identifying of candidate gene related to disease.2. The express level of eNOS and nNOS is down-regulated imply that retinal vascular endothelial cells and neurons of diabetic rat have been damaged. 3-NT- positive cells and more iNOS-positive cells appear in inner nucleus layer and ganglion cell layer imply that neural retina of 8-week diabetic rat has suffered from oxidative damage by NO, and has presented neuropathy.3. High glucose can damage diabetic retina and then make the expression of ET,ETRA,ETRB,ECE is up-regulated. The damage on neural retina is more seriously than on retinal blood vessels because the express level of ET,ETRA,ETRB is higher in inner nucleus than that in vascular layer.4. The higher express ofα-synuclein in ocular cone and ocular rod layer of 8-week diabetic retina imply that the photoreceptor cells of early diabetic retina have been damaged, neural retina has appear pathological changes.