The Effects Of Lipid On The Expressions Of TGF-β2 And TβRⅡ And The Apoptosis Of Retinal Endothelial Cells In Diabetic Retina

Research backgroundDiabetic retinopathy is one of the important cause of visual disability in diabetic subjects,its precisely mechanism is not yet fully understood, and recognized as the result of multiple factors synergistic action.Despite considerable progress in understanding of hyperglycemia-induced disease over the past decade,the link between lipid metabolic disorders and retinopathy still eludes us.Clinical observations have shown that there is a intimate link between lipid metabolic disorders and retinopathy,so it is important to investigate the mechanism by which lipid leads to diabetic retinopathy.Diabetic retinopathy is characterized by capillary occlusions, microaneurysms,selective loss of intramural pericytes,acellular capillaries,thickening of the basement membrane,and finally, angiogenesis and neovascularization.In the pathogenesis of retinopathy in diabetes,retinal microvascular endothelial,Muller cell,and ganglion cells and pericytes are lost selectively via apoptosis before other histopathology is detectable.It is not reported Whether dyslipidemia can induce the apoptosis of retinal endothelial cells.Neovascularization is strongly associated with retinal ischaemia, and growth factors have been implicated in its pathogenesis.The ischaemic retina is assumed to secrete growth factors which stimulate residual vessels to proliferate.The molecular mechanisms of dyslipidemia leading to diabetic retinopathy have not been studied in detail.Transforming growth factor beta(TGF-β) is involved in endothelial cell proliferation,adhesion,and deposition extracellular maxtrix.There has three well-defined mammalian isoforms,numerically designated as TGF-β1-3,eye tissue extensively expresses TGF-β, principally TGF-β2 in retina tissue.TGF-βacts as a bifunctional regulator of growth and it may either stimulate or inhibit proliferation depending on cell culture condition,TGF-βconcentration,or other cytokine.TGF-βis able to regulate angiogenesis by the regulation of endothelial cell growth and migration and the promotion of tube formation and vessel maturation.TGF-βexerts its biological effects by binding to specific cell surface receptors on target cells.Two cell surface TGF-βreceptor(TβRⅠ,TβRⅡ) proteins regulate the effects of TGF-βand the complex consisting of the typeⅠand typeⅡtransmembrane serine / threonine participates in signaling pathway and kinase activity of the typeⅡreceptor is essential in signaling pathway.There has no report about the effect of dyslipidemia on the expression of TGF-β2 and TGF-βtypeⅡreceptor in retina.According to the research background above,the present study was designed to investigate the effects of lipid on the expression of TGF-β2 and its receptor(typeⅡ) and the apoptosis of retinal endothelial cells in retina.Chapter 1.The effects of high fat diet ingestion on the expressions of retinal TGF-β2 and TβRⅡin streptozotocin-induced diabetic ratsObjective To investigate the effects of high fat diet ingestion on retinal TGF-β2 and TβRⅡmRNA in streptozotocin-induced diabetic rats.Methods After high fat diet was given to diabetic rats for 24 weeks,the expressions of TGF-β2 and TβRⅡmRNA in retina of diabetic rats were determined by RT-PCR analysis.Results The level of TGF-β2 mRNA in diabetic rats was increased compared with non-diabetic rats(P＜0.01);The level of TGF-β2 mRNA in diabetic rats on high fat diet was significantly increased compared with that of diabetic rats on normal fat diet(P＜0.01);The level of TβRⅡmRNA was no significant difference among diabetic rats and non-diabetic rats on high fat diet or normal fat diet.Conclusion High fat diet ingestion can increase TGF-β2 mRNA expression in retina of diabetic rats,and has no effect on the expression of TβRⅡmRNA in retina.Chapter 2.The effect of LPC on the apoptosis of bovine retinal endothelial cellsObjective To study whether LPC treatment can induce apoptosis of bovine retinal endothelial cells in vitro.Methods After the isolation of active retinal blood vessels,BRECs were cultured in the media of DMEM containing 20%fetal bovine serum and 5%human platelet-poor plasma and 20μl/ml retinal extract and were purified through separating and weeding for the colonies of cells in primary culture.BRECs were exposed to a rang of LPC concentrations(0-60 umol/L)and cultured in 6-well plates for 6,12,24 hours.Apoptosis was examined by acridine orange(AO) staining under fluorescence microscope.Flow cytometry was used to detect apoptosis rate.The intracellular content of calcium was determined with F-2000 type spectrofluorometer,and intracellular concentration of free calcium ion was calculated.Results BRECs with high purity can be obtained by using the media of DMEM contained 20 %fetal bovine serum,5%human platelet-poor plasma and 20μl/ml retinal extract and by separating and weeding for the colonies of cells in primary culture.After LPC treatment for 24 hours,the rate of apoptosis was significantly higher at LPC concentration 60umol/L compared to 20 or 40umol/L in bovine retinal endothelia cells.The concentration of free calcium ion was obviously elevated when the concentration of LPC from 20umol/L to 60umol/L,and the duration of LPC treatment had influence on concentration of free calcium ion,longer duration of LPC treatment had higher concerntration of free calcium ion.Conclusion Relative purity BRECs were attained and reproducible by using a selective cultural method.LPC treatment is able to increase the intracellular concentration of free calcium ion and to induce apoptosis of bovine retinal endothelial cells in a time and dose-dependment fashion.Chapter 3.The effects of LPC on the expressions of TGF-β2 and TβRⅡin bovine retinal endothelial cellsObjective To investigate the the effects of LPC on the expressions of TGF-β2 and TβRⅡin bovine retinal endothelial cells.Methods Bovine retinal endothelial cells were isolated from bovine eyes,cultured in vitro and exposed to a rang of LPC concentration(0-60umol/L).The levels of TGF-β2 and TβRⅡmRNA were determined by RT-PCR and heminested RT-PCR analysis,respectively.Results RT-PCR showed the level of TβRⅡmRNA was significantly higher at LPC concentration 40umol/L compared to 20 or 60umol/L after 12 hours.Heminested RT-PCR showed that the change of LPC concentrations had no significant effect on the expression of TβRⅡmRNA.Conclusion These results demonstrate that LPC regulates TGF-β2 mRNA expression and has no significant effect on the level of TβRⅡmRNA in bovine retinal endothelial cells.