【作者】 喻日成；

【导师】 雷闽湘；

【作者基本信息】 中南大学 ， 内科学， 2006， 博士

【摘要】 研究背景糖尿病视网膜病变（Diabetic Retinopathy,DR）是患者致盲的主要原因之一,其确切的发病机制尚不清楚,目前认为是多种因素协同作用的结果。在过去的几十年来对这种高葡萄糖导致的疾病的认识有很大的进展,但脂代谢紊乱在糖尿病微血管病变中的作用机制目前尚不清楚。临床观察表明脂代谢紊乱与糖尿病视网膜病变有密切关系,因此研究其作用机制就显得尤为重要。DR的病理改变包括毛细血管闭塞,微动脉瘤形成,血管壁内周细胞的选择性丧失,基底膜的增厚和新生血管形成。在DR中,视网膜微血管内皮细胞、Muller细胞、神经节细胞、周细胞通过凋亡而丧失,这种情况在DR的早期就可出现,目前国内外还未见有关脂代谢紊乱与视网膜内皮细胞凋亡关系的相关报道。新生血管形成与视网膜缺血关系密切,生长因子在其病理过程中起重要作用。缺血的视网膜能分泌生长因子,刺激残存的血管增殖。脂代谢紊乱导致DR的分子机制目前仍不清楚。TGF-β与内皮细胞的增殖、黏附和细胞外基质的聚积有关。在哺乳动物中,TGF-β有三种异构体,命名为TGF-β1、TGF-β2、TGF-γ3。TGF-β在眼组织中有广泛表达,其中在视网膜组织中主要表达TGF-β2。TGF-β作为一种具有双相功能的生长调节子,其刺激或抑制增殖的作用依赖细胞的培养条件、TGF-β的浓度及其他协同调节的细胞因子。TGF-β通过调节内皮细胞的增殖、移行,促进血管腔的形成和成熟而参与新生血管形成。TGF-β通过与靶细胞表面的特异受体结合而发挥生物效应。细胞有表面的二种TGF-β受体（TβRⅠ、TβRⅡ）,这二种受体的跨膜丝氨酸/苏氨酸部分通过形成受体复合物参与TGF-β的信号通路,从而调节TGF-β的效应,Ⅱ型受体的激酶活性是信号传导所必需的。目前还未见有关脂代谢紊乱对视网膜TGF-β2及其Ⅱ型受体影响的相关报道。基于上述原因,本研究探讨脂毒性对视网膜TGF-β2、TβRⅡ表达及内皮细胞凋亡的影响。第一章:高脂喂养对糖尿病大鼠视网膜TGF-β2与TβRⅡmRNA表达的影响目的:探讨高脂喂养的糖尿病大鼠视网膜中TGF-β2及TβRⅡmRNA的表达。方法:应用RT-PCR方法检测高脂喂养的糖尿病大鼠视网膜中TGF-β2及TβRⅡmRNA的表达水平。结果:糖尿病大鼠视网膜中TGF-β2 mRNA的表达高于非糖尿病大鼠视网膜（P＜0.01）;高脂喂养的糖尿病大鼠视网膜中TGF-β2 mRNA的表达高于普通喂养的糖尿病大鼠视网膜（P＜0.01）;TβRⅡmRNA的表达水平在非糖尿病大鼠视网膜与糖尿病大鼠视网膜间比较无明显差异;高脂喂养的糖尿病大鼠视网膜中TβRⅡmRNA的表达水平与普通喂养的糖尿病大鼠间比较亦无明显差异。结论:高脂喂养的糖尿病大鼠视网膜中TGF-β2 mRNA的表达水平升高。高脂喂养对糖尿病大鼠视网膜中TβRⅡmRNA的表达无影响。第二章溶血卵磷脂对牛视网膜微血管内皮细胞凋亡的影响目的:探讨溶血卵磷脂（LPC）对牛视网膜微血管内皮细胞（BRECs）凋亡的影响。方法:视网膜微血管消化分离后,采用含20%胎牛血清、5%PPP和20μl/ml视网膜浸出液的DMEM培养基,结合原代细胞的分离、除杂培养BRECs。培养的BRECs中加入不同浓度的LPC（0～60umol/L）,分别培养6小时、12小时、24小时,通过吖啶橙染色在荧光显微镜下观察细胞凋亡,流式细胞检测细胞凋亡率,F2000荧光分光光度计测细胞内钙离子浓度并计算游离钙浓度。结果:（1）含20%FBS的DMEM培养基中加入5%PPP及20μl/ml的视网膜浸出液,结合原代细胞克隆的分离、除杂,可获得较纯净的BRECs;（2）LPC浓度为60 umol/L作用BRECs 24小时后,BRECs凋亡率明显高于LPC浓度为20umol/L及40umol/L时;LPC浓度从20umol/L增加到60umol/L过程中,细胞内游离钙离子浓度明显增加,而且细胞内游离钙离子浓度的增加与LPC的作用时间有关,作用时间越长,钙离子浓度越高。结论:（1）通过选择性培养能获得高纯度的BRECs,并能连续传代;（2）LPC能增加细胞内游离钙离子浓度,促使BREC的凋亡,而且LPC的这种作用具有时间和剂量依赖性。第三章:LPC对牛视网膜微血管内皮细胞TGF-β2与TβRⅡmRNA表达的影响目的:探讨不同浓度的LPC对牛视网膜微血管内皮细胞TGF-β2与TβRⅡmRNA表达的影响。方法:原代分离、培养牛的视网膜微血管内皮细胞,加入不同浓度的LPC（0～60umol/L）,分别培养6小时、12小时、24小时,RT-PCR检测TGF-β₂ mRNA表达水平,半巢式RT-PCR检测TβRⅡmRNA的表达水平。结果:RT-PCR结果表明,LPC浓度为40umol/L作用12小时后,TGF-β2 mRNA水平明显高于LPC浓度为20 umol/L和60umol/L;LPC浓度改变对TβRⅡmRNA的表达无明显影响。结论:LPC能调节牛视网膜内皮细胞TGF-β2的表达;LPC对牛视网膜内皮细胞TβRⅡ的表达无明显影响;更多还原

【Abstract】 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.更多还原