【作者】 李世迎；

【导师】 阴正勤；

【作者基本信息】 第三军医大学 ， 眼科学， 2006， 博士

【摘要】 目的:通过将人胚胎视网膜神经上皮层和色素上皮层联合移植到光诱导视网膜变性猪的视网膜下腔,探讨大动物视网膜移植的方法,观察此类异种移植后的安全性、移植物和宿主的活体及离体形态学变化、以及宿主视功能的改变。并且探讨人胚胎视网膜的短期、中期及长期保存的适宜方法。从而为临床视网膜移植治疗视网膜变性疾病提供理论基础。方法:本研究首先用白色荧光灯持续照射6月以上,建立小型猪的光诱导视网膜变性模型。然后对12-35周健康流产人胚胎视网膜进行Ames’液、中期保存液(DX液)、负80度冰箱、深低温程序降温仪4种方法的保存,并通过台盼蓝染色、MTT比色来检测保存后细胞活性率,HE染色来评估其保存后结构。同时,选12-30周健康流产胎儿为供体,用准分子激光微切削脉络膜制备视网膜神经上皮层和色素上皮层(RPE)联合移植片。对14只光变性猪(28眼)和3只正常猪(5眼)共33眼行手术。光变性猪移植手术25眼,伪手术3眼;正常猪移植手术4眼,伪手术1眼。术后1周、1、2、3、5、8月行检眼镜、眼底彩照、荧光造影以及多焦视网膜电图(mfERG)和多焦视诱发电位(mfVEP)检查,并通过HE染色、免疫荧光,观察移植物在宿主视网膜下腔的存活能力和发育状况,移植物与受体视网膜的整合方式以及视网膜功能和视皮层功能的变化。结果:(1)、白色荧光灯光照6个月后,猪眼mfERG的1-6环各指标均比正常猪差,尤以1-2环振幅密度下降明显;mfVEP的P1波潜伏期在3环、5环比正常猪延长,电镜下见光变性猪视网膜光感受器层线粒体肿胀、胞浆溶解。(2)、人胚胎视网膜的Ames’液短期保存时间在4h内效果最佳,最长不应超过6h;DX液中期保存1-2天时效果较好;负80度冰箱中期保存1周时尚可;在长期保存1月时,深低温程序降温后的效果好于负80度冰箱。(3)、早期视网膜移植手术成功率66.67%。术后8月内行眼底彩照和荧光造影,发现移植区无荧光渗漏,有1只猪眼术后8月仍见移植区新生血管,1只猪眼术后5月仍见移植区内有荧光遮蔽。术后5月内可供取材10眼,均为光变性猪眼,在宿主视网膜下腔找到移植物7眼,移植物存活率为70%,包括3种情况:更多还原

【Abstract】 Purpose: To evaluate the safety, morphology and function changes of neuroretina-RPE cografts and host retina after transplanting sheets of human fetal neural retina together with its retinal pigment epithelium (RPE) to light-induced retinal degeneration mini-pigs. And to explore the proper ways to store fetal retina in short, middle and long term periods.Methods: Firstly model light-induced retinal degeneration mini-pigs by constant white light exposing 6 months.Then store12-35 weeks fetal retina in Ames’medium,DX medium, 80 centigrade below zero refrigerator and freeze control systems in different term periods respectively.Histology and cell viability were evaluated. 25 eyes of light-induced retinal degeneration mini-pigs and 4 normal eyes of minipigs underwent transplantation of 12-30 weeks human fetal neurortina-RPE cografts. 3 eyes of light-induced retinal degeneration and 1 normal eyes received false operations. In the follow-up of 1 week,1,2,3,5 and 8 months, the morphology of grafts and host were showed by ophthalmoscope, fundus colour graphy,fundus fluorescence angiography (FFA),histology and fluorescence- immunohistochemistry, the hosts’function changes were evaluated by multifocal electroretinogram (mfERG) and multifocal visual evoked potential (mfVEP).Results:(1) After 6 months constant white light exposing, mini-pigs’mfERG showed worse results compared with normal eyes, especially the P1 wave’s amplitude density in ring1 and 2. mfVEP displayed the prolonged latency of P1 wave in ring3 and 5. Electron microscopy showed swelled mitochondrion and lysised cytoplasm in photoreceptor layer.(2) In the storage of human fetal retina, Ames’medium showed best preservation in four hours, no longer than six hours. DX medium showed good preservation in one to two days. 80 centigrade below zero refrigerator could provide ordinary middle term preservation in one week. freeze control systems showed better preservation in one month period than 80 centigrade below zero refrigerator.(3) 23 eyes of the total 33 eyes(66.67%) underwent retinal transplantation successfully. In the 8 months follow-up, fundus fluorescence angiography showed no leak in fluorescein. In the survival period of 5 months,10 eyes specimens whose retina had been light- induced更多还原