【作者】 陈伟；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 眼科学， 2009， 博士

【摘要】 研究背景年龄相关性黄斑变性（age-related macular degeneration,ARMD）是一种老年常见病,流行病学调查发现其发病率越来越高,因ARMD致盲的人数将会超过因青光眼和糖尿病性视网膜病变的致盲数,成为一个社会性的公共问题。然而,现在临床上对已经发生损伤的ARMD患者治疗效果并不明显,TAP（Treatment of Age-RelatedMacular Degeneration with Photodynamic Therapy）和VIP（visualimpairment project）研究发现,在重度ARMD临床治疗组同未治疗组相比较分别仅仅有5%、8%的病人通过治疗视网膜下新生血管而获得视力提高。现在已经认识到对其研究的重点应放在预防ARMD的发生和发展上,包括预防干性ARMD中视网膜色素上皮细胞（retinalpigment epithelium,RPE）的氧化损伤和湿性ARMD中新生血管的生成,而不是单纯治疗各种已发生的损伤。近年来,自然药物在人类疾病的预防和治疗中应用越来越广,其中1983-1994年研究的520种药物中,39%是来自自然产品,1999年销售量最大的20种非蛋白类药物中,9种是由自然产品发展而来。在ARMD的防治上已经有些药物正崭露头角,如博视康牌叶黄素片等,但其昂贵的价格和其主要的抗氧化损伤作用,使其在应用上有一定的局限性。而delphinidin为花色素的一种主要成份,大量存在于蔬菜和水果中,已经研究其有强的抗氧化、抗新生血管生成、抗肿瘤生长的能力,其是否对RPE细胞的氧化损伤和脉络膜血管内皮细胞新生血管生成功能具有抑制作用目前不清楚。我们假定delphinidin对RPE细胞和内皮细胞的作用同对其他细胞一样,既可以抗氧化损伤,又可以抑制新生血管生成,则对其的研究有望为ARMD的防治带来新的曙光。目的1探讨delphinidin对ARPE-19细胞氧化损伤的保护作用及其机制。2研究delphinidin对缺氧环境下ARPE-19细胞和猴脉络膜视网膜血管内皮（hesus choroids-retina endothelial cell line,RF/6A）细胞VEGF表达的影响及可能机制。3观察delphinidin抗RF/6A细胞体外新生血管生成的作用及机制。方法1利用MTT比色法,观察体外培养条件下,delphinidin对ARPE-19细胞增殖活性的影响;Hoechst染色、JC-1、ROS试剂盒和RT-PCR分别检测细胞在H₂O₂氧化损伤下不同浓度delphinidin（1μM、5μM、10μM、20μM）预处理组同对照组中细胞凋亡、线粒体胞膜电位、ROS含量和bcl-2 mRNA含量的变化。2传代培养ARPE-19、RF/6A细胞,Ⅷ因子免疫组化鉴定RF/6A细胞;MTT检测delphinidin对RF/6A细胞毒性;delphinidin（1μM、5μM、10μM、20μM）预处理组和对照组采用western-blot法检测各组细胞在CoCl₂损伤下对VEGF、HIF-1α、HIF-2α蛋白表达的变化。3传代培养RF/6A细胞,实验分为对照组及delphinidin（1μM、5μM、10μM、20μM）预处理组,采用MTT和caspase-3免疫组化法检测各组在VEGF刺激下RF/6A细胞增殖和凋亡情况;transwell小室迁移和Matrigel胶体外血管形成实验观察细胞迁移及血管形成情况;western-blot检测各组VEGF刺激下P-FAK蛋白表达变化。结果1.10^-6M～10^-1M delphinidin处理ARPE-19细胞后未发现有细胞毒性。用1μM、5μM、10μM、20μM delphinidin分别预处理RPE细胞后进行H₂O₂氧化损伤,发现凋亡细胞的数量减少,线粒体膜电位的降低程度减少,细胞内ROS含量减少,bcl-2 mRNA含量增加,且这些变化具有剂量依赖性,各处理组与对照组相比差异有显著性,组间比较差异有统计学意义（P＜0.05）。2.Ⅷ因子胞浆着染证明所用RF/6A细胞为内皮细胞,10^-6M～10^-1M delphinidin并未显示出对RF/6A细胞毒性。1μM、5μM、10μM、20μM delphinidin预处理RPE、RF/6A细胞组可见CoCl₂损伤后的VEGF、HIF-1α蛋白表达较对照组明显减少,且呈剂量相关性,各处理组与对照组相比差异有显著性,组间比较差异有统计学意义（P＜0.05）。而HIF-2α蛋白的表达并未受到影响,各处理组同对照组相比较差异均无统计学意义（P＞0.05）。3.1μM、5μM、10μM、20μM delphinidin预处理RF/6A细胞组可呈剂量依赖性抑制VEGF刺激下细胞的体外增殖、迁移和体外血管形成,同时P-FAK蛋白表达降低,各处理组与对照组相比差异有显著性,组间比较差异有统计学意义（P＜0.05）。结论:1 Delphinidin可减轻H₂O₂造成的ARPE-19细胞氧化损伤,其机制可能是通过改善线粒体的功能,减少凋亡发挥作用,并呈浓度依赖性。2 Delphinidin可以抑制缺氧状态下ARPE-19和RF/6A细胞VEGF的表达,此过程是通过减少HIF-1α蛋白的表达来发挥作用,与HIF-2α蛋白无关。3 Delphinidin可以抑制VEGF刺激下RF/6A细胞体外新生血管生成功能,其作用机制可能与抑制FAK的磷酸化激活有关。更多还原

【Abstract】 BACKGROUNDWith the aging of the population,age-related macular degeneration （ARMD） is becoming a public health concern and there will be more elderly persons who blind because of ARMD than glaucoma and diabetic retinopathy.The excitement associated with the advances in antiangiogenic therapy for patients with ARMD has been tempered by the clinical findings that after successful regression of subretinal neovascularization,visual improvement has been limited.In both the TAP （Treatment of ARMD with Photodynamic Therapy） and the VIP （Verteporfin in Photodynamic Therapy） studies,only 5%and 8%of patients,respectively,gained vision compared with control subjects after the treatment of subretinal neovascularization.As a result,it is becoming increasingly clear that new approaches for treatment should be focused on both preventing the initial insults that lead to disease progression and rescuing the retinal pigment epithelium（ARPE-19） and photoreceptor cells that have been damaged.Nowdays medicine of food has become more and more popular in prevention and treatment of many diseases.Of the 520 new drugs approved between 1983 and 1994,39%were natural products,of which 60—80%were antibiotics and anti-cancer drugs from natural products.Of the 20 best-selling non-protein drugs in 1999,9 were derived or developed as the result of leads generated by natural products.Some medicine have come into being such as ocuvite for preventing ARMD,but the expensive price and mainly function for anti-oxidant has limit it’s applying.Delphinidin as one part of anthocyanidin,has many effect for anti-oxidant,anti-angiogenicanti-carcinosis and anti-inflammatory properties which may be helpful for the prevention and treatment of many diseases,but has not been elucidate could the effect to antioxidant on RPE cells and inhibited the angiogenesis on RF/6A（rhesus choroidsretina endothelial cell line）cells.We think delphinidin have these functions on RPE and RF/6A cells,then we can find an effective prevention for ARMD.OBJECTIVE1)To investigate the effect of delphinidin on anti-oxidative stress injury and the relative mechanisms in ARPE-19.2) To investigate the efect of delphinidin on expression of VEGF （vascular endothelial growth factor）and the relative mechanisms in RPE and RF/6A cells induced by hypoxia mimic cobalt chloride.3) To investigate the effect of delphinidin on angiogenesis and the relative mechanisms in cultured RF/6A cells.METHODS1) MTT was used to determine the effect of delphinidin on ARPE-19 cell survival and proliferation at a series of concentration.Cells was pretreated with delphinidin（1,5,10,20μM ） for 24 hours then under H₂O₂ induced-stress-injury.The morphodifferentiation of apoptotic nuclei was analyzed by Hoechst-PI fluorescence staining.The concentration of ROS and JC-1 was measured by ROS and JC-1 kit.The mRNA expression of bcl-2 was measured by RT-PCR.2) The primary cultured RF/6A cells was identified by immunohistochemical method.MTT was used to determine the effect of delphinidin on RF/6A cell survival and proliferation at a series of concentration.The RPE and RF/6A cells was pretreated with delphinidin（1,5,10,20μM ）for 24 hours then under COCl₂ induced-hypoxic condition.The expression of VEGF,HIF-1αand HIF-2αprotein were measured by Western-blot.3) Cells was pretreated with delphinidin（1,5,10,20μM ） for 24 hours then under VEGF-stimulating.The cell proliferation was assessed using MTT after 24 hours.Cell migration was investigated by transwell chamber and tube formation on matrigel by endothelial cells was also analyzed.The protein expression of p-FAK and cell apoptosis was measured by western-blot and caspase-3 immunohistochemical method. manner between the concentration of 1μM～20μM.As compared with negativ group,bcl-2 mRNA were significantly up-regulated in 5μM～20μM delphinidin pretreated group（P＜0.01）,while in 1μM delphinidin pretreated group（P＜0.05）.2) Immunohistochemical method identificated RF/6A cell was endothelial cell.Delphinidin at the concentration of 10^-6M～10^-1M didn’t inhibit the proliferation of RF/6A cells in vitro.While delphinidin inhibits VEGF and HIF-1αprotein in a dose-dependent manner.As compared with negativ group,VEGF and HIF-1αprotein were significantly down-regulated in 5μM～20μM delphinidin pretreated group（P＜0.01） and in 1μM delphinidin pretreated group（P＜0.05）,while HIF-2αprotein didn’t change compared with negativ group（P＞0.05）.3) Delphinidin inhibits VRGF-stimuated RA/6A cell proliferation, migration、tube formation and protein expression of p-FAK in a dose-dependent manner.As compared with negativ group,cell proliferation,migration,tube formation and protein expression of p-FAK were significantly down-regulated in 5μM～20μM delphinidin pretreated group（P＜0.01）,while in 1μM delphinidin pretreated group（P＜0.05）. Caspase-3 immunohistochemical show delphinidin inhibit cell proliferation was not through apoptosis.CONCLUSION1) Delphinidin exerts a potent anti-oxidative effect in vitro and the relative mechanism may be associated with the inhibition of the function of mitochondrion.2) Delphinidin could inhibit the expression of VEGF under hypoxic condition,and the relative mechanism may be associated with the inhibition of HIF-1α,while not associated with HIF-2α.3) Delphinidin exerts a potent antiangiogenic effect in vitro and the relative mechanism may be associated with the inhibition of p-FAK.更多还原