| Diabetic retinopathy (DR) is the main complication of diabetes mellitus (DM), and also is one of the most serious diseases in ophthalmology. It remains a leading cause of vision loss and new-onset blindness. DR is a progressive microangiopathy characterized by small vessel damage and occlusion. In DR patients the time of onset and therefore the duration of disease are more difficult to determine precisely. Recent studies show that cytokines are involved in the pathogenesis of DR in general. Along with the technological development in molecular biology, it has been discovered that the occurrence and development of DR is related to the abnormal regulation of cell proliferation, and several cytokines and growth factors are closely related to the growth and proliferation of several retinal cells, among which transforming growth factor-β (TGF-β) is an important factor. TGF-β is a multifunctional cytokine, whose numerous cell and tissue activities include cell-cycle control, the regulation of early development, differentiation, proliferation, migration, extracellular matrix formation, hematopoesis, angiogenesis, chemotaxis, immune functions, and the induction of apoptosis. All these functions are transduced through the TGF-β signaling pathway. TGF-β transduces signals through the mediation of its specific receptors. There are two major types of receptors,TGF-β type I (TβR I) and TGF-β type II (TβR II )transmembrane serine/threonine kinase receptors, are believed to be the signaling molecules. There is , however, little information on process of TGF-β receptors induction in retina of Diabetic rats. In this study,we choose TβR I and TβR II genes as the target genes, to quantitatively detect the expression level of TβR I and TβR II genes in the retina of normal rats in order to determine the expression difference of TβR I and TβR II in retina, which will be able to provide the technical and substantial foundation for the study of gene in retina, we alsoinvestigated the expression of TpR I and T0RII in different stage of Diabetic rats' retina with real-time fluorescence quantitative reverse transcription polymerase chain reaction (QRT-PCR), and discussed the possible functions of TpR I and TPR II in the pathogenesis of diabetic retinopathy, and provided experiment data and experience for the further clinic study.Part 1. The detection of TpR I and TpR II expression in retina of normal ratsObjective : To quantitatively detect gene expression level of TpR I and TpR II inretina of normal rats.Methods: 10 Sprague-Dawley(SD) rats(male, approximately 200g) were used.Theanimals' retinas were dissected .The total RNA was preparation.Results: The RNA of rat retina was integrative enough to be used for furtherQRT-PCR analysis. The mRNA level of TpR I/18s were 0.00034+0.00013. ThemRNA level of TpR II /18s were 0.0001 ±0.00005 in retina of normal rats. The meanration of TpR I / TpRlI were 3.9±1.7(P <0.001).Conclusions: QRT-PCR could specifically and accurately detect gene expressionlevel in rat retina. The method is practical and feasible. The expression of TGF-Preceptors in normal rat retinas suggested that TpR I and TpR II played an importantrole in the homeostasis of normal retina. It will be able to provide the preliminarytechnical and substantial foundation for the study of gene in retina.Part 2. Quantitative detection of TpR I and TpR II expression in retina ofDiabetic rats.Objectives: To quantitatively detect gene expression level of TpR I and TpR II in different stage of diabetic rats' retina to observe and analyze the effect of TGF-P receptors on the retina of rat diabetic animal model.Methods: 28 healthy adult Sprague-Dawley rats were chosen and randomly divided into two groups of normal contrast(CON) and diabetes mellitus(DM). Diabetic rats were induced by streptozotocin (STZ) injection intraperitoneally. Gene expression was detected quantitatively with QRT-PCR.Results: The mRNA level of T(3R I and TpRlIwas 0.000493±0.000133 and 0.000166±0.000057 at 4weeks. The mRNA level of TpR I and TpR II was 0.000608±0.000232 and 0.000113±0.000049 at 12weeks. TpR I expression was gradually elevated during the progression of diabetic retinopathy, TpR II expression was up-regulated at 4weeks, but down-regulated at 12weeks.Conclusion: TGF-P and its receptors (TpR I and TpR II) may play important role in the pathogenesis of diabetic retinopathy.
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