【作者】 田润；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 眼科学， 2009， 博士

【摘要】 第一章缺氧对ARPE-19细胞表达TGF-β2、VEGF、PEDF及TSP-1的影响目的:探讨缺氧诱导人视网膜色素上皮（retinal pigment epithelial,RPE）细胞株（ARPE-19）表达转化生长因子—β2（transforming growthfactor-β2,TGF-β2）、血管内皮生长因子（vascular endothelial growthfactor,VEGF）、色素上皮衍生因子（pigment epithelium-derived factor,PEDF）及血小板反应蛋白—1（Thrombospondin-1 TSP-1）的情况。方法:含10%胎牛血清的DMEM/F12培养基培养ARPE-19细胞,加入150umol/mlCoCl₂进行化学缺氧,细胞免疫荧光染色及WesternBlot技术检测ARPE-19细胞在缺氧0h、4h、8h、12h、24h、48h,TGF-β2、VEGF、PEDF及TSP-1的表达情况。结果:1.细胞免疫荧光染色ARPE-19细胞胞浆表达VEGF蛋白,随缺氧时间的延长表达增强,以缺氧24小时最为显著;ARPE-19细胞胞浆表达PEDF蛋白,随缺氧时间的延长表达减弱;ARPE-19细胞胞浆、胞膜、核膜及核仁均有TSP-1及TGF-β2蛋白的表达,随缺氧时间的延长表达减弱。2.Western Blot技术检测ARPE-19细胞缺氧组表达VEGF随时间延长表达较常氧对照组明显增加,以24小时达高峰,缺氧组为常氧组的2.6倍,此后有所下降,缺氧与常氧组除0小时外（P=0.935）,其它各时间点差异均有统计学意义（P=0.000-0.002）;表达PEDF、TSP-1及TGF-β2缺氧组较常氧对照组明显减少,48小时缺氧组分别为常氧组的0.30倍、0.35倍及0.28倍,缺氧与常氧组除0小时外（P=0.125）、（P=0.803）、（P=0.086）,其它各时间点差异均有统计学意义（P=0.000-0.014）、（P=0.000-0.003）、（P=0.000-0.001）。3.缺氧不同作用时间APRE-19表达PEDF、TGF-β2与TSP-1成正相关（r=0.902,P=0.000）、（r=0.990,P=0.000）;缺氧不同作用时间APRE-19表达VEGF与TSP-1成负相关（r=-0.853,P=0.000）。结论:1.缺氧启动血管新生可能通过ARPE-19细胞表达促血管生成因子VEGF的增加及抗血管生成因子PEDF、TSP-1及TGF-β2的减少实现。2.TSP-1不同于作为血管生成正向作用因子的VEGF,可能与PEDF及TGF-β2一同作为血管生成的负向作用因子参与血管新生性疾病的发生发展。第二章VR-10合成多肽对恒河猴脉络膜视网膜内皮（RF/6A）细胞的作用目的:探讨VR-10合成多肽对猴脉络膜视网膜内皮细胞株（RF/6A细胞）增生、移行的作用及对TGF-β2、VEGF、PEDF表达的影响。方法:MTT法检测1μg/ml外源性TSP-1及0.1μg/ml、1μg/ml及10μg/ml的VR-10合成多肽在作用6、12、24、48小时后对RF/6A细胞增殖的影响;Transwell小室实验检测作用24小时后对细胞移行的影响;细胞免疫荧光染色及RT-PCR技术检测对RF/6A细胞表达TGF-β2、VEGF、PEDF的影响。结果:1.MTT法检测不同浓度和时间TSP-1及VR-10合成多肽作用RF/6A细胞存活率:TSP-1及VR-10合成多肽对RF/6A细胞均有抑制作用,且随作用时间及浓度的增加,细胞存活率越低。以作用48小时VR-10合成多肽10μg/ml组细胞存活率最低为78%（P＜0.001）。2.Transwell小室实验中TSP-1及VR-10合成多肽对RF/6A细胞迁移具有抑制作用（P＜0.001）;以10μg/ml的VR-10合成多肽抑制率最高,1μg/ml TSP-1全肽抑制率次之。VR-10合成多肽随浓度增加抑制率越高（P＜0.001）;0.1μug/ml的VR-10合成多肽与1μg/ml的VR-10合成多肽对RF/6A细胞的抑制率差异无明显统计学意义（P=0.114）。3.细胞免疫荧光染色RF/6A细胞胞浆表达TGF-β2蛋白,1μg/mlTSP-1处理RF/6A细胞表达TGF-β2较空白对照组强,各浓度VR-10合成多肽处理RF/6A细胞表达TGF-β2与空白对照组比较无明显差别;RF/6A细胞胞浆表达PEDF蛋白,TSP-1及VR-10合成多肽处理RF/6A细胞表达PEDF均较空白对照组表达增强,以10μg/ml浓度组VR-10合成多肽作用最强;RF/6A细胞胞浆表达VEGF蛋白,TSP-1及VR-10合成多肽处理RF/6A细胞表达VEGF均较空白对照组表达减弱,以10μug/ml浓度组VR-10合成多肽作用最强。4、RT-PCR检测除1μg/ml浓度TSP-1组处理RF/6A细胞表达TGF-β2 mRNA较空白对照组表达增强外（P=0.000）,其余各组均与空白对照组间比较差异无统计学意义（P＞0.05）;PEDF mRNA均较空白对照组表达增强,以10μg/ml浓度组VR-10合成多肽作用最强（P＜0.001）,各组间比较差异有统计学意义（P＜0.001）:VEGF mRNA均较空白对照组表达减弱,以10ug/ml浓度组VR-10合成多肽作用最强（P＜0.001）,除1μg/ml浓度组VR-10合成多肽与1μg/mlTSP-1多肽组间比较差异无统计学意义外（P=0.615）,其余各组间比较差异有统计学意义（P＜0.001）。结论:1.VR-10合成多肽具有抑制内皮细胞增殖、移行的作用。2.VR-10合成多肽通过上调抗血管生成因子PEDF,下调促血管生成因子VEGF的表达,及TGF-β2非依赖机制,共同作用而抑制血管新生。第三章VR-10合成多肽对RF/6A细胞表达Fas/FasL、Caspase-3及Bcl-2的影响目的:探讨VR-10合成多肽对RF/6A细胞凋亡相关基因表达的影响。方法:RT-PCR技术检测10μg/ml的VR-10合成多肽及空白对照组对RF/6A细胞表达Bcl-2及FasL mRNA的影响。Western Blot技术检测10μg/ml的VR-10合成多肽及空白对照组对RF/6A细胞表达Fas及Caspase-3蛋白的影响。结果:1.RT-PCR检测RF/6A细胞表达Bcl-2 mRNA,10μg/mlVR-10合成多肽处理组较空白对照组表达减少（P=0.000）,RF/6A细胞表达FasL mRNA,10μg/mlVR-10合成多肽处理组较空白对照组表达增强（P=0.001）。2.WB检测空白对照组RF/6A细胞表达Caspase-3蛋白大多为无活性酶原形式（32KD）;10μg/ml VR-10合成多肽处理RF/6A细胞主要表达Caspase-3活性小分子片段（20KD）;10μg/ml VR-10合成多肽处理RF/6A细胞表达Fas蛋白较空白对照组增加（P=0.000）。结论:VR-10合成多肽通过增加凋亡促进基因Fas/FasL而活化Caspase-3,同时伴有生存基因Bcl-2的减少,共同作用介导内皮细胞凋亡。更多还原

【Abstract】 Part 1 Expression of cell factors by human retinal pigment epithelial（RPE） cell（APRE-19） under hypoxic conditionPurpose:To Investigate the production and relaease of transforming growth factor-β2（TGF-β2）,vascular endothelial growth factor（VEGF） pigment epithelium-derived factor（PEDF）and thrombospondin-1（TSP-1） by human retinal pigment epithe（?）（RPE） cell（APRE-19） under hypoxic condition.Methods:APRE-19 cells were cultured in DMEM/F12 medium with 10%fetal calf serum.Using 150μmol/ml CoCl₂ to simulate the hypoxic condition.After 0,4,8,12,24 and 48 hours of hypoxia,TSP-1, TGF-β2,VEGF and PEDF peptides were detected by immunofluorescent staining and Western Blot.Results:1.Immunostaining for VEGF was observed in the cytoplasm of ARPE-19 cells,and it showed a time-dependent increase by hypoxia.The most remarkable expression was at 24 hour of hypoxia. Immunostaining of PEDF was observed in the cytoplasm of the ARPE-19 cells,and it showed a time-dependent derease by hypoxia. Immunostaining of TGF-β2 and TSP-1 was observed in the cytoplasm, entoblast,epicyte,and karyotheca of the ARPE-19 cells,and they showed a time-dependent derease by hypoxia.2 Western Blot identified a time-dependent increase of VEGF peptides in the media of hypoxic ARPE-19 cells compared to normoxic cells.At 24 hour of hypoxia,the expression of VEGF reached the peak,it was 2.6 times more than that of normoxic condition,and then decresed. There were signifieanfly statistical differences between hypoxic group and normoxic group at each time（P=0.000-0.002） excluding at 0 hour （P=0.935）.Western Blot identified a time-dependent decrease of PEDF, TSP-1 and TGF-β2 peptides in the media of hypoxic ARPE-19 cells compared to normoxic cells.At 48 hour,the expression of PEDF,TSP-1 and TGF-β2 peptides in hypoxic groups were normoxic groups’ 0.30 times,0.35 times and 0.28 times respectively.There were signifieantly statistical differences between hypoxic group and normoxic group at each time（P=0.000-0.014）,（P=0.000-0.003）,（P=0.000-0.001） excluding at 0 hour（P=0.125）、（P=0.803）、（P=0.086）3.Positive correlations（r=0.902,P=0.000）、（r=0.990,P=0.000） were observed between the expression of PEDF,TGF-β2 and TSP-1.A negative correlation（r=-0.853,P=0.000） was observed between VEGF and TSP-1.Conelutions:1.Hypoxia boot up neovascularization by the increase of angiogenic growth factor VEGF and the decrease of anti-angiogenic facors PEDF,TGF-β2 and TSP-1.2.TSP-1 was different from positive role of VEGF,it might contribute to the happening and development of the disease by the negative role similar with PEDF and TGF-β2. Part 2 Role of synthetical peptide VR-10 on rhesus choroidal -retinal endothelial（RF/6A） cell.Purpose:To investigate the effects of synthetical peptide VR-10 on proliferation and migration of rhesus choroidal -retinal endothelial （RF/6A） cell and the expressions of TGF-β2,VEGF and PEDF in RF/6A cell.Methods:Proliferation of RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10（0.1μg/ml,1μg/ml and 10μg/ml） after 6h,12h,24h and 48h were detected by the tetrazolium dye-reduction assay（MTT）.Transwell chamber was used to investigate the migration of RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10（0.1μg/ml,1μg/ml and 10μg/ml） after 24h.The expression of mRNA of TGF-β2,VEGF and PEDF in RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10（0.1μg/ml,1μg/ml and 10μg/ml） were detected by immunofluorescent staining and reverse transcription -polymerase chain reaction（RT-PCR） analysis.Results:1.TSP-1（1μg/ml） and synthetical peptide VR-10 （0.1μg/ml,1μg/ml and 10μg/ml） inhibited proliefration of RF/6A cells in a time and dose-dependent way.Survival ratio of RF/6A was decrease with the increase of time and concentration.The lowest survival ratio of RF/6A was 78%（P＜0.001） by the treatment of 10μg/ml synthetical peptide VR- 10 after 48h.2.TSP-1 and synthetical peptide VR-10 could inhibite migration of RF/6A cells in transwell chamber（P＜0.001 ）.10μg/ml synthetical peptide VR-10 had the strongest effect,1μg/ml TSP-1 was the next.Migration inhibition rate was increase with the increase of the concentration of synthetical peptide VR-10（P＜0.001）.There was no signifieantly statistical differences between 0.1μug/ml synthetical peptide VR-10 and 1μg/ml synthetical peptide VR-10（P=0.114 ）.3.Immunostaining for TGF-132 was observed in the cytoplasm of RF/6A cells,the expression was increase after exposure to 1μg/ml TSP-1 compared with control group.There were no impact on the expression of TGF-β2 after exposure to synthetical peptide VR-10.Immunostaining of PEDF was observed in the cytoplasm of the RF/6A cells,and the expressions were increased aider exposure to 1μg/ml TSP-1 and synthetical peptide VR-10 compared with control group.10μg/ml synthetical peptide VR-10 had the strongest effect.Immunostaining of VEGF was observed in the cytoplasm of the RF/6A cells,and it decrease after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10 compared with control group.10μg/ml synthetical peptide VR-10 had the strongest effect.4 Expression of TGF-β2 mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1（P=0.000）.There were no signifieantly statistical differences between synthetical peptide VR-10 and control group（P＞0.05）.Expression of PEDF mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1 and synthetical peptide VR-10, and 10μg/ml synthetical peptide VR-10 had the strongest effect（P＜0.001）. There were significantly statistical differences between groups（P＜0.001）. Expression of TGF-β2 mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1（P=0.000）.There were no significantly statistical differences between synthetical peptide VR-10 and control group（P＞0.05）.Expression of PEDF mRNA in RF/6A cell was decreased after treatment of 1μg/ml TSP-1 and synthetical peptide VR-10, and 10μg/ml synthetical peptide VR-10 had the strongest effect（P＜0.001）. There were significantly statistical differences between groups（P＜0.001）, excluding 1μg/ml synthetical peptide VR-10 and 1μg/ml synthetical peptide VR-10（P=0.615）.Conclusions:1.Synthetical peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell.2.Synthetical peptide VR-10 had anti-angiogenic ability by up-regulation of anti-angiogenic factor PEDF,down-regulation of angiogenic factor VEGF and TGF-β2 independent mechanism. Part 3 Role of Synthetical Peptide VR-10 on the Expression of Fas/FasL、Caspase-3 and Bcl-2 by RF/6A CellPurpose:To investigat the influence of synthetical peptide VR-10 on the expressions of apoptosis relative genes in RF/6A cell.Methods:The expression of Bcl-2 and FasL mRNA in RF/6A cell after exposure to 10μg/ml synthetical peptide VR-10 were detected by RT-PCR analysis.The expression of Fas and Caspase-3 peptides in RF/6A cell after exposure to 10μg/ml synthetical peptide VR-10 were detected by Western Blot.Results:1.Compared with control group,expression of FasL mRNA were significantly increased in 10μg/ml synthetical peptide VR-10 treated group,but the expression of Bcl-2 mRNA was decreased.2.Western bolt showed that RF/6A cell in control group mainly expressed the 32-kD procaspase-3 forms.To 10μg/ml synthetical peptide VR-10 treated group,it showed decreased expression of procaspase-3（32 KD） and concomitant increased expression of its shorter proapoptotic forms（20 KD）.Compared with control group,expression of Fas peptides were significantly increase in 10μg/ml synthetical peptide VR-10 treated group.Conclusions:Synthetical peptide VR-10 mediated endothelial cell apoptosis and inhibited angiogenesis in association with increased expression of Fas/FasL,decreased expression of Bcl-2,and processing of caspase-3 into smaller proapoptotic forms.更多还原