Proteomics Study Of Neural Retinas From Type 2 Diabetic Rats

Diabetic retinopathy(DR), a common chronic complication ofdiabetes, is one of leading causes of blindness and visual disability inelderly worldwide. The data indicate that the prevalence rate of DR isabout 50.0% in diabetes. Approximately 10% of patients with diabetes formore than 15 years have developed severe visual impairment, and about2% have become blind.Although the pathological process and correlative pathologicmechanism of DR have been widely studied by using conventionalmethods, the biochemical and molecular pathways that underlie theinitiation and progression of diabetic retinopathy remain unresolved andthere is no effective means to prevent and cure DR even today.Proteomics, a new approach to study of diseases, is not to study ofindividual protein as traditionally, but rather to study the entirecomplement of proteins expressed by a cell or tissue at a given time.The multiple factors and the complex regulatory network that are responsible for pathological process of DR can be revealed by comparingthe protein expression profiles from normal tissue to those frompathological tissue, and also the cellular and molecular mechanism of DRbe unraveled.In the first section of the present study, neural retinas were dissectedfrom eyes of Sprague-Dawley (SD) rats and separated neural retinas wereidentified by histopathology and transmission electron microscope (TEM).Total proteins of neural retina were extracted and high-resolution 2DEwas used to separate total proteins. The experiments were repeated tooptimize the experiment conditions of the isoelectric focusing (IEF),SDS polyacrylamide gel electrophoresis(SDS-PAGE), silver staining(or coomassie-blue-staining) and 2DE techniques. Well resolved,reproducible 2DE profiles of rat neural retinas were establised. Theresults provide a basis for further research on the diabeticretinopathy.In section two, normal age-matched control SD rats and 8 weeksdiabetic rats induced by feeding a high-fat(HF) diet combined withintraperitoneal(i.p.) injection of streptozotocin (STZ) were researched.Total proteins were extracted from neural retinas of two groups of rats,then high-resolution 2DE was used to separate total proteins followed bysilver staining or coomassie-blue-staining. Computer assisted imageanalysis(Image Master 5.0) to quantitative compare and analyze the differences in protein expression between age-matched control rats anddiabetic rats. Total 3122±37(n=3) spots were detected in normal samplesand 2702±21(n=3) spots in diabetic rats. The average matching rate was76.8%. Most protein spots were visualized within a pI range of 4.0-8.0and a molecular mass range from 14.4 to 66.4 kDa. It was found byimage analysis that about 150 spots in 2DE gel of diabetic retinas thatexhibited statistically significant variations, including up-regulation of 68proteins in diabetic rat retinas, down-regulation of 82 compared withnormal rat retinas. Twenty differentially expressed proteins wereidentified by peptide mass fingerprinting (PMF) or peptide sequence, inwhich ten proteins were newly discovered proteins associated with DR.The functions of identified proteins were primary analyzed. Thedifferentially expressed proteins revealed by our experiments werediverse, including proteins involved in saccharide and lipid metabolism,inflammatory response, nitric oxide synthetase pathway, as well ascytoskeletal and molecular chaperone proteins.Then, the proteinαA-crystallin was chosen as a target for specificimmunodetection using western blot. Moreover, immunofluorescence(IMF) staining was carried out to demonstrate the expression and locationofαA-crystallin in the retina. The resultes corroborate the variation foundby 2DE.A histological method(HE staining)was used to detect the neuronal degeneration of diabetic retina and found the decreased density ofganglion cell in the retinas from diabetes group.In short, using a proteomics approach we separated and identifiedproteins differentially expressed in the diabetic retinas and find somespots were probably novel proteins. Western blot and immunofluore-scence staining were used to corroborate the variation found by 2DE. Ahistologic method demonstrated neuronal degeneration of diabetic retinain the early stage of diabetes. The methods applied in present study werestable and the results were reliable. All data provided a new understand tothe pathomechanism of the DR.