重组色素上皮衍生因子对氧诱导大鼠视网膜新生血管生长的抑制作用

  本文关键词：重组色素上皮衍生因子对氧诱导大鼠视网膜新生血管生长的抑制作用，，由笔耕文化传播整理发布。

        研究背景早产儿视网膜病变(retinopathy of prematurity, ROP)已经成为全世界范围内致盲的重要原因,主要由于未成熟儿视网膜发育不完善,在多种因素影响下视网膜缺血,进一步造成视网膜新生血管形成,然后导致增殖性视网膜病变。视网膜新生血管形成后可发生渗出,出血,增殖膜等一系列病变,使眼球的结构和功能受到严重的损害ROP的治疗主要是激光光凝术和冷冻手术,晚期的患者行玻璃体切割术,手术治疗虽然可控制ROP的病程,防止严重并发症,但治疗效果仍不理想,只是延缓病变的进展,在治疗过程中破坏了视网膜组织,导致视功能的永久性损害。ROP的发生与视网膜新生血管有密切关系。但新生血管增殖的机制不明,新生血管的形成是一及其复杂的生物过程。研究发现血管内皮生长因为与视网膜新生血管形成密切相关,视网膜新生血管性疾病均存在这视网膜组织血管内皮生长因子(vascular endothelial growth factor, VEGF)生成增加。而抑制视网膜组织VEGF的表达,能够使视网膜新生血管形成减少。VEGF作为。种血管生成刺激因子被认为在ROP血管形成中起到重要的中心调控作用,血色素上皮衍生因子(pigment epithelium-derived factor, PEDF)是VEGF的非特异性抑制剂。两者在视网膜血管病变中有着密切的关系。在缺氧的视网膜中,PEDF(?)的含量会下降,VEGF(?)的含量增多。PEDF的产生和分布是很广泛的,成人眼内的PEDF存在于角膜,房水,玻璃体,视网膜等,主要由视网膜色素上皮分泌而来。PEDF不仅能够影响视网膜分化,发育和成熟,还有神经元保护作用,具有神经营养和抗新生血管的作用。PEDF是目前最强大的新生血管再生抑制因子之一。另外它只针对异常新生血管而不抑制正常存在血管,而且,PEDF的神经营养和保护作用,对比可能损害神经的抑制药物或生物因子来说,更占优势。因而PEDF具有广阔的临床应用前景。PEDF还具有抗氧化作用,这些因素都是早产儿视网膜病变的主要因素,所以可以想象PEDF在预防和治疗早产儿视网膜病变的新生血管形成及进展方面的作用。因此,我们的实验通过不同的氧气浓度建立合适的氧诱导视网膜病变(oxygen induced retinopathy, OIR)模型,模拟人类未成熟儿视网膜病变,观察VEGF与视网膜新生血管之间的关系,采取PEDF两种不同的注射方式(结膜下、玻璃体腔)治疗,观察PEDF对视网膜新生血管的抑制作用及对正常视网膜血管的影响,力图寻找一种简便、安全、有效的途径,为未成熟儿视网膜病变药物治疗提供新的方法和思路。目的观察不同氧浓度环境在新生鼠视网膜病的作用,建立符合早产儿视网膜病变病理特点的动物模型,为研究该病的发病机制及治疗提供实验基础,利用视网膜血管ADP酶染色及组织切片观察大鼠视网膜形态及病变程度。分析新生鼠视网膜及氧诱导视网膜病变中VEGF蛋白质表达的变化。方法将新生鼠40只,与母鼠共同饲养,将其分为空气组；波动组1:50%-20%；波动组2：40%-10%；波动组3:50%-10%四组,每组各10只。正常空气组正常空气中饲养18d,波动组1,放入50%0224h,20%24h,循环七次,共14d,后放入空气饲养4d,波动组240%0224h,10%24h,循环七次,共14d,后放入空气饲养4d,波动组3放入50%0224h,10%24h,循环七次,共14d,后放入空气饲养4d。18的时,每组各取6只大鼠左眼,行视网膜铺片ADP酶染色观察视网膜血管形态的发育,钟点法评价视网膜新生血管严重程度；每组各取6只大鼠右眼,制作眼球病理切片,常规HE染色计数突破视网膜内界膜的内皮细胞核定量分析视网膜新生血管增生情况,比较各组间的差异。各组剩余视网膜组织,利用Western-blot检测视网膜血管内皮生长因子VEGF蛋白的表达。统计分析：所有数据均依据SPSS13.0统计软件包进行分析,实验测试指标的数据资料以x±s表示；采用One-Way ANOVA分析数据,方差齐时组间采用LSD法,方差不齐时组间多重比较采用Dunnett C法,P<0.05作为差异有显著性意义。结果视网膜铺片ADP酶染色：正常空气组新生鼠在18d时视网膜血管发育基本成熟,管径较粗,全视网膜分深浅两层,浅层自视盘发出,向四周呈放射状均匀分布,深层呈多角形网状形态；波动组1初网膜摁管发育基本正常；波动组2,3视网膜血管扩张迂曲,中央见无灌注区,血管发育未达视网膜周边部,有大量新生血管形成,血管密度增高,新生血管网结构紊乱,有新生血管丛和血管瘤。四组钟点法评价新生血管钟点数为：0,0.17±0.41,4.8±1.2,7.8±1.2,不同吸氧浓度导致的视网膜新生血管钟点数比较,差异有显著性意义(F=120.21,P=0.00)；空气组波动组2,3病变钟点数明显增多,与相应的正常空气组相比较,差异有统计学意义(P=0.00)；波动组1无明显增多,与正常空气组比较差异无统计学意义；波动组3比波动组2相比较增多,相比有统计学意义(P=0.00)。HE染色：四组突破内界膜内皮细胞核数分别为0.6±0.470.87±0.6514.28±3.8322.52±4.02,四组之间比较差异有显著性意义(F=875.366,P＝0.00);正常空气组鼠几乎没有见到突破视网膜内界膜的血管内皮细胞核,波动组1切片中绝大多数视网膜内界膜是平滑的,偶见新生血.管。波动组2,3组所有切片均可见较多突破内界膜的血管内皮细胞,所有切片均可见较多突破内界膜的血管内皮细胞,且波动3组突破内界膜的血管内皮细胞明显比波动2组多(P<0.01)；VEGF表达在所有波动组中,与波动组1,2比较,波动组3中VEGF表达最高,空气组表达最低。结论1.50%-10%氧气波动组最适合制作早产儿视网膜病变模型,具有类似ROP血管闭塞,血管无灌注区,新生血管等改变,制备过程较为简便,重复性好,是研究ROP比较适合的动物模型；2.证实了反复血氧浓度波动可导致新生鼠视网膜新生血管增生性病变,吸入氧分压差(ΔFiO2)的波动与病变程度呈正相关；3.低氧比高氧对发生视网膜病变可能更重要；4.VEGF在视网膜新生血管形成中起着重要作用,促进视网膜新生血管形成。目的观察结膜下和玻璃体注射重组人色素上皮衍生因子(pigment epithelium-derived factor, PEDF)两种注射方式对氧诱导大鼠视网膜新生血管(Retinal neovascularization, RNV)的作用,从治疗角度阐述PEDF在ROP中的作用。方法新生大鼠分别进行氧气或空气暴露：新生鼠与哺乳母鼠置于密闭容器内,50%0：24h,10%/L0224h为1个循环,共7个循环,建立类似早产儿视网膜病变动物模型。氧诱导SD新生大鼠建立类似早产儿视网膜病变动物模型。新生鼠48只随机分为6组(A：空气对照组,B:高氧对照组,C:高氧+玻璃体注射PEDF2ug组,D：高氧+结膜下注射PEDF2ug组,E：高氧+结膜下注射PEDF4ug组,F：高氧+结膜下注射PEDF8ug组)。当新生大鼠脱离氧时,C,D,E和F组大鼠左眼用不同剂量和注药次数进行注射PEDF,共四天,18d处死取标本,ADP酶视网膜血管染色观察视网膜血管形态,石蜡切片计数突破视网膜内界膜的新生血管内皮细胞核数目。另新生大鼠2只进行左眼结膜下注射PEDF, Western-blot检测视网膜PEDF的表达.统计分析：所有数据均依据SPSS13.0统计软件包进行分析,实验测试指标的数据资料以x±s表示；采用One-Way ANOVA分析数据,方差齐时组间比较采用Bonferroni法,方差不齐时组间多重比较采用Dunnett C法,P<0.05作为差异有显著性意义。P<0.05为差异有统计学意义。结果结膜下注射PEDF,可检测到视网膜PEDF蛋白表达：视网膜铺片结果：,A组视网膜血管发育正常。B组视网膜大量的新生血管生成。C组新生血管明显减少。D、E、F组新生血管稍减少。六组组织病理学检测突破视网膜内界膜的新生血管内皮细胞计数分别为：1.88±0.92,25.00±0.94,12.23±0.96,24.58±1.03,23.68±0.77,23.20±0.88,各组比较差异有统计意义(F=6729,P=0.00);A组视网膜内界膜平滑,偶见突破的视网膜内皮细胞。B组明显高于A组(p=0.00),差异有统计学意义。C组明显低于高氧埘照组B组(p=0.00),D组与B组相比无明显差异(p=0.20),E、F组突破内界膜的内皮细胞核数’与C组差异有统计学意义(p=0.00)。结论1、结膜下注射PEDF,巩膜和脉络膜-色素上皮层对其是有渗透性的,可以跨越结膜下组织到达视网膜,但与玻璃体注射组相比,抑制新生血管作用有明显差异；2、玻璃体注射PEDF可以有效抑制氧诱导大鼠视网膜新生血管；3、结膜下注射PEDF如何有效到达视网膜还需进一步研究。

    BackgroundROP is proliferative disease of the retinal vasculature infants that may cause severe visual loss, and it is a major cause of blindness in newborn, The disease is characterized by proliferation of abnormal fibrovascular tissue at the border of vascularised and non-vascularised retina. Retinopathy of prematurity (ROP) is a potenitially blinding disease affecting premature infants. The immature neurovascular tissues of the visual system, the retina and brain, are particularly susceptible to injury. ROP is characterized by pathological ocular angiogenesis or retinal neovasculrization (NV).This NV leads to traction retinal or further proliferation of fibrovascular tissue on the retinal surface, resulting in formation of a retrolental fibrovascular membrane, total retinal detachment, intravitreal hemorrhages and subsequent vision loss. The approved treatments consist in photocoagulation surgery, laser photocoagulation or cryotherapy. However, the visual outcomes after treatment often are not satisfactory. While guidelines for the management and treatment of ROP have recently been refined, it is still difficult to identify those infants in whom disease is more likely to progress. Preventive therapy for ROP is sorely needed.Abnormal development of the retinal vasculature is at the heart of this disease. Vascular endothelial growth factor(VEGF) has emerged as one of the most important in the development of NV. PEDF is a member of the serine protease inhibitor (serpin) family, although it does not have protease inhibitory activity. Pigment epithelium-derived factor(PEDF) is the most potent natural inhibitor of angiogenesis. PEDF has emerged as a potentially important endogenous inhibitor of ocular neovascularization. PEDF is a multifunctional serpin protein. It is PEDF also has neuroprotective activity and has been demonstrated to protect photoreceptors from degeneration. PEDF has been found in the vitreous and cornea, and it has been found to be downregulated by hypoxia in cultured cells. Its presence in the vitreous and its antiangiogenic activity suggest a possible role for PEDF in the regulation of retinal neovascularization. PEDF strongly inhibited VEGF-induced migration and proliferation of retinal endothelial cells.Therefore, our experiment used oxygen concentration variation to establish a suitable oxygen-induced retinopathy rat model for imitating human ROP. We analysed the relationship between VEGF and retinal neovascularization, We observed the effects of PEDF on OIR in rats with subconjunctival or intravitreal injection. We strived a novel, safer and more effective therapeutic approaches for the treatment of pathologic neovascular conditions.BackgroundRetinopathy of prematurity (ROP) is a causing-blindness eye disease Oxygen in premature retinal neovascularization plays an important role, but its scope of oxygen and the specific mechanism of retinopathy is not clear ObjectiveTo study the effects of different concentrations of oxygen on neovascularization in oxygen-induced retinopathy (OIR) of newborn rats and provide experimental basis for clinical oxygen therapy; To determine the roles of vascular endothelial growth factor (VEGF)in vascularization of the developing retina.Method:Forty Sprague-Dawley rats were included in the study. They were divided into four groups:Air group (10rats) was was raised in air room. Fluctation group1、2、3(10rats) was exposed to cycles of50%～20%,40%～10%,50%～10%for14days, respectively. In each case, oxygen was alternated between the two exposure concentrations every24hours. The rats were removed to room air for4days before the severity of abnormal neovascularization were measured. Retinas were dissected and stained by adenosine-diphosphatase(ADPase) histochemistry for assessmem of intraretinal vascular development and preretinal angiogenic vessel growth. The proliferative neovascularization response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. The total number of nuclei counted per retina was defined as the nucleus count. Nuclei above the internal limiting membrane were counted. Protein expressions of VEGF were studied by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±tandard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the LSD method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.Result Retina flat-mounts (ADP-ase staining):The retinal vascularization had completed in normal air group, The main buanches of vessels in optic idsc are well-developed in normal air group. Retina vascular development is normal basically;Retina flat-mounts presented increasing neovascular tufts,plenty of newly born vascular plexus and disorderly arranged vessel structures in Fluctation group2and Fluctation group3.The clock number of these four groups were0,0.17±0.41,4.8±1.2,7.8±1.2,respectively. the difference was significant (F=120.21, p=0.00). The difference was statistically significant between Fluctation group2and Fluctation group3(P=0.00).HE stain results:The number of vascular nuclei extending from the internal limiting membrane into the vitreous were0.6±0.47,0.87±0.65,14.28±3.8322.52±4.02, respectively. The difference was significant (F=875.366, p=0.00). The differences in the VEGF-expression among three groups were significant (P=0.00).Conclusion1.50%～10%pattern is suitable for oxygen-induced retinopathy rat model to imitate human ROP; This animal model(50%～10%pattern) of OIR is reproducible. Because of its quantifiable characteristic, it is useful for the study of pathogenesis of retinal neovascularization and theraputic intervention for ROP.2. Changeable Oxygen concentrationa could trigger a subsequent neovascularization;△FiO2were positively correlated with the retinopathy;3. Hypoxia may be more important for retinal neovascularization on the occurrence of retinopathy than Hyperoxia;4. The over-expression of VEGF plays a important role during the formation of neovascularization. ObjectiveTo observe the inhibitive effect of intravitreal or Subconjunctival injecting PEDF for retinal neovascularization in Oxygen-induced animal models.MethodMouse models of oxygen-induced retinopathy of prematurity were established.48newborn rats were randomly diveded into6groups (A: Air control group; B:Hyperoxygen control guoup; C:Hyperoxygen+intravitreal injection of2ugPEDFgroup; D:Hyperoxygen+injection of2ugPEDFgroup; E:Hyperoxygen+injection of4ugPEDFgroup; F:Hyperoxygen+injection of8ugPEDFgroup). When the rats were taken off the high oxygen, the PBS and corresponding dosage of PEDF were intravitreal or Subconjunctival injected in group C, D, E and F, respectively. ADP-ase stained retina flat-mounts was used to evaluate the retinal vascular modality and development, Eyeball pathological section was used to count the pre-retinal endothelial nucleus in retinal new vessel. Retinal PEDF expression were detected by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the Bonferroni method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.ResultsPEDF were achieved in retinal after a periocular injection. Retinal flat-mounts: A group:The retina vessels of the normal had a fine radial branching pattern that extend from the optic nerve to the periphery; B group:There were more retinal neovascularization compared with A group.The density and shape of retinal neovascularization were disordered; Comparing with B group, retinal neovascularization decreased obviously in C group while D, E, F group decreased mildly. HE staining:A group:Retinal internal limiting membrane was smooth, and occasionally the endothelial cells break through the inner retinal. B group was significantly more than group A(p<0.05). C group was significantly less than the B group (p<0.05), D, E, F group had significant difference between group C (all p <0.05).Conclusion1. PEDF are able to penetrate the sclera into retinal after a periocular injection, however, compared with intravitreal injection, the effect reduced significantly.2. Intravitreal injection of PEDF can arrest retinal neovascularization in SD rat models;3. How to reach the retina effectively requires further study by subconjunctival injection.

        重组色素上皮衍生因子对氧诱导大鼠视网膜新生血管生长的抑制作用

摘要3-8ABSTRACT8-13前言15-21    参考文献17-21第一部分 不同吸氧浓度诱导视网膜病变大鼠模型21-45    背景内容21    1 材料21-24    2 实验方法24-28    3 结果28-35    4 讨论35-41    5 参考文献41-45第二部分 PEDF抑制ROP新生血管形成的实验研究45-61    1 材料45    2 实验方法45-48    3 结果48-54    4 讨论54-58    5 参考文献58-61全文总结与展望61-62附录一 缩略词表62-63攻读学位期间的研究成果63-64致谢64-66统计学审稿证明66

  本文关键词：重组色素上皮衍生因子对氧诱导大鼠视网膜新生血管生长的抑制作用，由笔耕文化传播整理发布。