人胚胎视网膜色素上皮细胞在传代中的衰老及其机制  

Mechanism of the aging phenomenon with passage of human retinal pigment epithelial cell

作　　者：赵颖[1] 牛膺筠[2] 

机构地区：[1]青岛大学医学院附属市立医院眼科,266011 [2]青岛大学医学院附属医院眼科,266003

出　　处：《中华实验眼科杂志》2012年第1期33-36,共4页Chinese Journal Of Experimental Ophthalmology

基　　金：基金项目：国家自然科学基金项目（30572010）

摘　　要：背景视网膜色素上皮（RPE）细胞的衰老直接影响视网膜光感受器细胞的代谢功能，导致视网膜功能的异常和视力丧失。深入研究RPE细胞的衰老机制将有助于研究年龄相关性黄斑变性（AMD）的预防和治疗。目的研究人胚胎RPE细胞在传代过程中的衰老并探讨其机制。方法收集胎龄16～28周的人胎儿眼球6个，对人胚胎RPE细胞进行分离、培养和传代以制作RPE细胞复制性衰老模型，选择3～12代的RPE细胞用于实验。对培养和传代的RPE细胞用免疫组织化学法以人角蛋白对细胞进行鉴定。应用四甲基偶氮唑蓝（MTT）比色法检测各代细胞的增生活力，以RPE细胞的吸光度（A490）表示。采用流式细胞仪检测传代细胞周期和细胞线粒体跨膜电位（△ψm）的变化。结果培养和传代的细胞呈六角形，人角蛋白检测呈棕色，第3代以后的细胞色素消失，3～6代的细胞形态正常，生长活跃，之后生长能力和增生活力下降。培养第3～12代RPE细胞随着传代的增加，处于G0／G1期的细胞百分比逐渐增加，差异有统计学意义（F=180．430，P=0．000），由第3代细胞的68．40％增加到第12代细胞的87．33％，细胞生长趋于停滞。各代间处于G0／G1期细胞的百分比比较，第6代细胞明显高于第3代，第9代明显高于第6代，第12代则明显高于第9代，差异均有统计学意义（t=4．002，P〈0．05；t=12．885，P〈0．01；t=16．387，P〈0．01）。MTT法对RPE细胞活力的检测结果表明，随着传代次数的增多，反映RPE细胞活力的A490值逐渐下降，总体比较差异有统计学意义（F=38．770，P=0．000），第9代、第12代RPE细胞的A490值分别明显低于第6代和第9代，差异均有统计学意义（t=5．991、11．983，P〈0．01）。同时，3—12代PRE细胞随着传代的增加，罗丹明123染色荧光强度逐渐降低，差异有统计学意义（F=121．680，P=0．000），�Background Retinal pigment epithelial （RPE） cell senescence damage the metabolism of photoreeeptor,leading to retinal dysfunction and loss of vision. To understand RPE cell senescence mechanism will contribute to the study of age-related maeular degeneration （AMD）. Objective The present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms. Methods This study was approved by the Ethic Committee of Qingdao University Medical College, and the informed consent was obtained from each gravida. Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks. RPE ceils were isolated,cultured and passaged in vitro to establish the cell replicative aging model. The third to twelfth cells were collected to be used to this experiment. Human keratin was used to identify the cells by immunochemistry, and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value. The cellular cycles and transmembrane potential （△ψm） of mitoehondrion （△ψm） with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin. The melanin was absent in the third generation cells. Vibrant growth statues were seen from the 3-6 generations cells and thereafterthe proliferation ability reduced. The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40% in the third generation of cells to 87.33% in the twelfth generation of ceils, showing a significant difference among various generations （ F = 180.43, P = 0. 00 ） , and that of the sixth, ninth and twelfth generation of cells was significant higher than the third, sixth and ninth generation respectively （t = 4. 002, P〈0. 05 ; t= 12. 885 ,P〈0.01 ;t= 16. 387 ,P〈0. 01 ）. MTT assay showed that of RPE cells we

关 键 词：视网膜/色素上皮 细胞衰老 细胞周期 线粒体 膜电位 

分 类 号：R774.1[医药卫生—眼科]