永生化胚胎视网膜干细胞的体外诱导分化  

Induced differentiation of immortal retinal stem cell in vitro

作　　者：由德勃[1] 俞海燕[1] 王薇[1] 

机构地区：[1]北京大学第三医院眼科中心,北京100083

出　　处：《眼科》2005年第1期51-54,i001,共5页Ophthalmology in China

基　　金：国家自然科学基金资助项目 (30 170 999);北京市科委重点基金资助项目 (H0 2 0 2 2 0 0 10 390 )

摘　　要：目的 :研究R718B2永生化视网膜干细胞系细胞的体外诱导分化。方法 :对R718B2细胞系细胞进行体外培养扩增 ,利用含有表皮生长因子 (epidermalgrowthfactor,EGF)、碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)及白血病抑制因子 (leukemiainhibitoryfactor,LIF)等生长因子的DMEM/F12培养液对第 4 3代细胞进行体外诱导分化 ,免疫荧光细胞化学鉴定诱导后细胞的抗原表达。结果 :诱导后的R718B2细胞系细胞增殖速度减慢 ,细胞表达微管相关蛋白 2 (micro tubule associatedprotein 2 ,MAP2 )、胶质源性纤维酸性蛋白 (glialfibrillaryacidprotein ,GFAP)、蛋白激酶C α(proteinkinaseC α ,PKC α)、Thy1 1及Calretinin ,同时部分细胞仍表达巢蛋白 (Nestin) ,但未见视紫红质 (Rhodopsin)表达。结论 :R718B2细胞是一种多能视网膜干细胞。在EGF及bFGF等多种生长因子的共同作用下 ,该细胞系中部分细胞仍能表达未分化细胞标记物Nestin ,部分细胞开始向神经元、神经胶质细胞及双极细胞、神经节细胞和无长突细胞等视网膜终末细胞方向分化。objective:to investigate the differentiation potential of immortalized r718b2 retinal stem cells in vitro.methods:r718b2,a human immortalized retinal stem cell line,was prelabeled with the green fluorescent protein (gfp).the cell line was maintained in dmem/f12 with 5% fbs.the 43th generation of this cell line was cultured in the conditioned medium with egf,fgf,and lif,but without fbs.at the end of 5 days in culture,the cells were harvested and incubated with specific antibodies (nestin,map2,gfap,rhodopsin,pkc-α,calretinin,thy 1.1) for immunocytochemical staining.result:the r718b2 cells expanded rapidly in the medium with 5%fbs.while when they were cultured in the medium with the growth factors,their expandition ability reduced obviously.in addition to nestin,the neuroectodermal stem cell marker,the cells that were exposed to the growth factors for 5 days expressed map2 ,gfap and several cell-type specific retina markers (pkc-α,calretinin,thy 1.1).but staining of rhodopsin,the marker of rod photoreceptor,was not detected.conclusion:our studies suggest that the r718b2 cells can survive well in conditioned medium with egf,bfgf and lif.some of them maintain their characters as a type of undifferentiated stem cell,whereas the others begin to differentiate into the terminal retina cell types including bipolar cell,amacrine cell,müller cell and ganglion cell.these evidences together with our previous research result strongly suggest that the r718b2 cell has the character of multipotential retinal stem cell.

关 键 词：视网膜 干细胞 分化 细胞培养 

分 类 号：R774[医药卫生—眼科]