| Chapter one The expression and its meaning of stromal cell-derived factor-1αand chemotaxis cytokine receptor-4 in diabetic retinopathy and in diabetic cardiomyopathyObjective:To study the expression and its meaning of stromal cell-derived factor-1α(SDF-1α)and chemotaxis cytokine receptor-4 (CXCR-4)in diabetic retinopathy and in diabetic cardiomyopathy.Methods:Adult Sprague-Dawley male rats,which weight was 200±20 gram,were randomly divided into diabetic group(DM)and control group(NC).Each group was divided into five subgroups,(2 weeks(2wk),4 weeks(4wk),8 weeks(8wk),12 weeks(12wk),20 weeks (20wk)respectively).Each subgroup contains 8 rats.Diabetes mellitus rat model was established with a single intraperitoneal injection of streptozotocin(STZ),control group was injected with a single intraperitoneal injection of equal sodium citrate buffer.Rat's weight,fasting plasma glucose(FPG)were measured before they were executed.The level of plasma SDF-1αwas detected by ELISA.The expression of SDF-1α,CXCR-4,VEGF mRNA in retina and heart were detected by RT-PCR.After perfusion by PBS and polyoxymethylene,the protein expression of SDF-1α,CXCR-4,VEGF in retina and heart were tested by immunohistochemistry.The morphology of retinal vascular was observed by H-E staining and transmission electron microscope(TEM).The changes of myocardium microvessel density(MVD)were observed by vWF staining.The protein expression of SDF-1α,CXCR-4,VEGF in retina and heart were also measured using Western blot.Results:(1)All diabetic rats were made successfully by a single intraperitoneal injection of streptozotocin(STZ).Compared with NC group,the FPG,the quantity of eating,drinking water and urine volume increased,while the weight of rats increased significantly slow(p＜0.01). (2)The level of peripheral blood SDF-1αof rats in DM group started increasing at 4wk,and it was higher significantly since 8wk compared with NC group(p＜0.01).The level of plasma SDF-1αincreased with the prolongation of disease in DM group(p＜0.05),but it had no apparent change with the prolongation of disease in NC group(p＞0.05).(3)In retinal micrangium of DM rats,the number of pericyte decreased but the number of acelluar vessels increased.The MVD decreased in DM group. The ultrastructure changes of pericyte and endotheliocyte could be found in DM group.(4)Compared with NC group,the mRNA and protein expression of SDF-1α,CXCR-4 and VEGF in retina of DM rats increased with the prolongation of disease(P＜0.05),and all got to peak at 20wk. But all of above index had no apparent change with time in NC group(P＞0.05).(5)Compared with NC group,the mRNA and protein expression of SDF-1αand CXCR-4 in heart of DM rats increased with the prolongation of disease(P＜0.05),and all got to peak at 20wk.But the mRNA and protein expression of VEGF did not markedly increase in all DM group.All of above index had no apparent change with time in NC group(P＞0.05).Conclusions:The expression of SDF-1α,CXCR-4 and VEGF in DM rats' retina are increased than NC rats'.The expression of SDF-1α, CXCR-4 in DM rats' heart are increased than that in NC rats',but the expression of VEGF is not increased markedly.Maybe the expression of SDF-1α,CXCR-4 and VEGF are related to the development of diabetic retinopathy.The different expressing of VEGF in retina and heart is concerned with diabetic paradox of poor vessel growth in ischemic heart and increased angiogenesis in ischemic induced retinal complications. Chapter two The effects of stromal cell-derived factor-1αon the expression of CXCR-4 and VEGF in bovine retinal endothelial cellObjective:To evaluate the effects and its mechanism of stromal cell-derived factor-1α(SDF-1α)on the expression of chemotaxis cytokine receptor-4(CXCR-4)and Vascular endothelial growth factor(VEGF)in bovine retinal microvascular endothelial cell(BREC)in vitro.Methods:The suspension of 3rd to 7th generation BREC was inoculated into 12 holes plate.Under common cultural condition,BREC was interfered with glucose,SDF-1αand glucose+SDF-1αin different concentration.Supernatant was obtained to detect the level of VEGF by ELISA and cells were obtained to detect the expression of CXCR-4 by Western blot.Results:(1)Normal glucose(5.6mM)did not affect the expression of CXCR-4 and VEGF.High glucose(25mM)stimulated bovine retinal endothelial cells(BRECs)express CXCR-4 and VEGF in a timedependent manner.(2)SDF-1αpromoted BRECs express VEGF and CXCR-4 in a dose-and time-dependent manner,and its effect achieved peak at the concentration of 100ng/ml,the level of CXCR-4 increased higher than VEGF did.(3)Both high glucose and SDF-1αcould stimulate BRECs to express VEGF and CXCR-4 in a synergetic manner,and the increasing level of CXCR-4 increased higher than that VEGF did.Conclusions:High glucose and SDF-1αstimulate BRECs express CXCR-4 and VEGF.Moreover,they stimulate BREC express CXCR-4 and VEGF synergistically.
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