【摘要】目的:探讨色素上皮衍生因子(pigment epithelium derived factor,PEDF)对光损伤的视网膜感光细胞的作用。
方法:Sprague Dawley(SD)鼠右眼玻璃体腔内注射PEDF,左眼注射磷酸盐缓冲液(phosphate buffered solution,PBS)作对照。注射2d后将SD鼠置于1000~1400lx 弥散白色冷荧光灯下连续光照2,5,7d后分别行ERG检查和组织学分析。
结果:连续光照2,5,7d后,PEDF处理组与PBS对照组的外核层(outer nuclear layer,ONL)厚度及ERG b波振幅均呈逐渐下降趋势,统计学分析两组差别具有显著性意义(P<0.01)。
结论:PEDF 对光损伤的视网膜感光细胞具有保护作用。
【关键词】 色素上皮衍生因子;光损伤;视网膜变性;感光细胞营救;视网膜电图
Study of pigment epithelium derived factor protecting photoreceptors from light damage in vivo
ShuXing Ji, Mi Yan, JunJun Zhang
Foundation items: National Natural Science Foundation of China (No.30400487);
Department of Science and Technology of Guangdong ProvinceKey GuidanceInternational Science and Technology Cooperation Project (No.2004B50301002; No.2004B50301002); "1135" Excellent Talents Project (20082010) of Daping Hospital of the Third Military Medical University, Chongqing, China
1 Department of Ophthalmology, Daping Hospital, the Third Military Medical University, Chongqing 400042, China; 2 Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Correspondence to: ShuXing Ji. Department of Ophthalmology, the Daping Hospital, the Third Military Medical University, Chongqing 400042, China. [email protected]
AbstractAIM: To explore the effect of pigment epitheliumderived factor (PEDF) on the retinal photoreceptor cells of rats damaged by bright continuous light (BCL). METHODS: Five to eightweekold albino SpragueDawley (SD) rats received an intravitreal injection of PEDF in right eyes. Fellow eyes received phosphatebuffered saline (PBS) and served as control group. Two days after the injection, the SD rats were exposed to the diffuse cool white light of 10001400lx for different periods(2, 5, 7 days). ERG responses were recorded after BCL damage. The ocular tissues were then processed for histological examinations.RESULTS: After 2, 5, 7 days BCL exposure, the thickness of the outer nuclear layer (ONL) and ERG responses all decreased in two groups, and there were significant differences between the mice in control and PEDF treatment group.CONCLUSION: PEDF protect photoreceptor cells function and morphology from BCL damage. KEYWORDS: pigment epithelium derived factor; light damage; retinal degeneration; photoreceptor rescue; electroretinographic response
0引言
在视网膜色素变性、老年型黄斑变性和视网膜脱离等许多致盲眼病中,感光细胞的死亡是不可逆转的。研究表明局部注射生长因子可以提高感光细胞的存活率[1]。色素上皮衍生因子(pigment epithelium derived factor,PEDF)是第一个由视网膜色素上皮(retinal pigment epithelium,RPE)细胞衍生的蛋白质,它是一种多功能的神经营养因子,能够诱导原代培养的Y79视网膜母细胞出现广泛的神经元样突起并分化[2]。本实验采用局部注射外源性PEDF的方法,观察其对光损伤的SD鼠视网膜感光细胞是否具有保护作用,以期更有效的治疗视网膜变性疾病。
1 材料和方法
1.1材料
65只SD鼠,5~8wk龄,体质量170~200g,雌雄不拘,由华西医学实验动物中心提供。实验分为PEDF组(30只)、磷酸盐缓冲液(PBS)组(30只)和无光照损伤组(5只),其中PEDF组和PBS组为自身同体对照。
1.2方法
1.2 .1 PEDF玻璃体腔内注射
为简化操作,人为规定每只SD鼠右眼为PEDF处理眼,左眼为PBS对照眼。右眼玻璃体腔内注入浓度为1g/L[3] 的PEDF液1μL(美国bioproducts公司产品)。左眼注射1μL PBS液。注射完毕后,直接检眼镜下观察有无视网膜脱离、玻璃体或视网膜出血及白内障形成,如有,则弃之不用。所有玻璃体腔内注射操作均在解剖显微镜下进行,注射2d后开始接受光照。
1.2.2光损伤模型的建立
参照谢伯林等[4] 建立动物视网膜光损伤模型的方法。采用40W弥散白色冷荧光灯(上海嘉定甲马灯泡厂,波长510~560nm),光照箱内各方向光照强度为1000~1400lx,平均1200.0±101.1lx(LX101 digital lux meter),以确保每只SD鼠充分接受光照。SD鼠在暗房内适应新环境10d后,开始进入光照箱内接受光照。分别于连续光照2,5,7d后检测ERG和组织学形态。
1.2.3 ERG记录
分别于连续光照2,5,7d后,记录SD鼠双眼的明、暗视ERG。散瞳及腹腔麻醉后,固定动物,甲基纤维素保持角膜湿润。记录电极为自制角膜接触镜电极,参考电极置于双眼颞侧皮下,地电极置于后肢皮下。暗视ERG刺激条件:采用全视野闪光刺激仪(美国Nicolet Ganzfeld产品)刺激时间0.1ms,敏感度500μV,放大倍数4000倍,刺激光强度0.5log cd/m2,对8个反应进行平均。明视ERG刺激条件:带宽1~1000kHz,背景光15Ftl,余刺激参数同暗室ERG刺激条件。记录b波振幅(Vbmax),规定b波振幅为a波和b波波峰之差,以便于定量分析。
1.2.4视网膜形态学观察及外核层厚度测定
分别于连续光照2,5,7d后,摘取眼球,于3点钟处留少许结膜作标记,40g/L甲醛溶液固定,常规石蜡包埋。经视乳头颞侧旁开1mm沿垂直子午线切成5μm厚的切片,显微镜下观察、摄片。采用美国Nikon & spot图像分析系统测量ONL厚度,作为感光细胞丧失的评判指标[4]。 统计学处理:数据以±s 表示,进行t检验分析,所用统计软件为SPSS 11.0,P<0.05为差别具有统计学意义。
2结果
2.1 ERG 正常SD鼠ERG波形由相对较小的a波和显著的b波以及振荡电位组成。连续光照2,5,7d后,在PBS组和PEDF组,暗视蓝光、暗视白光和明视白光ERG b波振幅均逐渐下降,各组间b波振幅差别均有显著性意义(P<0.01,图1,表1)。表1SD鼠暗视蓝光、白光和明视白光ERG b波振幅(略)注: *代表数值太低,测不出;bP<0.01 vs PBS对照组
2.2组织学
视网膜光损伤的主要部位是光感受器外节,表现为肿胀,极性消失,空泡变性,感受器内节细胞核固缩,RPE细胞屏障功能破坏,其中损害严重者,感受器和RPE细胞消失,代之以Müller细胞和其它胶质细胞,ONL的厚度也变薄(图2,表2)。表2SD鼠各组ONL厚度比较(略)
3讨论 PEDF是由Tombran 等[2]首先在胚胎RPE细胞的条件培养基中发现的具有神经营养作用的蛋白质,其基因位于染色体17p13.1[5,6],与视网膜色素变性常染色体显性位点密切相连。它由RPE细胞分泌产生,贮存于感光细胞基质内[7]。研究表明PEDF在视网膜神经元培养系统能够抑制过氧化氢诱导的细胞凋亡[8];在遗传性视网膜变性鼠模型中可以延长感光细胞的存活时间[3];在去除RPE细胞后,外源性PEDF可以维持正常感光细胞神经元的发育和视紫质的表达[9]。Yabe等[10]证实NF kappaB是PEDF发挥其神经保护作用所必需的信号转导分子,PEDF 通过此途径来维持小脑颗粒细胞神经元的存活,从而诱导其它抗凋亡和/或神经营养因子基因的表达。 我们将外源性PEDF注射入SD鼠玻璃体腔内,连续光照后进行ERG和组织形态学检查,结果表明PEDF组暗视蓝光、暗视白光和明视白光ERG b波振幅与PBS组的相应 b波振幅差别均具有显著性意义,组织形态学结果表明PEDF组的鼠眼ONL得到了较好的保留,从而从功能学和解剖形态学上证实PEDF具有保护受连续光损伤感光细胞活性的作用,同时也表明PEDF可能是在感光细胞受损伤的早期阶段发挥其保护作用。PEDF对锥体细胞的保护作用对维持明视功能特别重要。Sygawara等[11]认为在光损伤鼠模型(1000~2000lx光照条件下),锥体细胞的丧失是杆体细胞丧失间接造成的,因鼠杆体细胞丧失50%多以后锥体细胞的丧失才明显,因此在PEDF组,锥体细胞的挽救可能是杆体细胞保存的间接结果。 目前研究已表明PEDF能够抑制碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的促有丝分裂和促血管生成活性[12,13],是一种非常有效的抗血管生成因子。PEDF对视网膜感光细胞神经元的保护作用以及它的抗血管生成的双重特性[14],必将为视网膜变性疾病的预防和治疗开辟一个全新的领域。其具体作用机制仍有待于进一步研究。
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