胎儿视网膜色素上皮细胞的原代培养和体外增殖能力的测定  

Characterization of human fetal retinal pigment epithelial cells in culture: proliferation rate and marker expression

作　　者：袁松涛[1] 薛志刚[2,3] 林卿[4] 刘庆淮[1] 范国平[5] 

机构地区：[1]江苏省人民医院眼科,南京210029 [2]同济大学苏州研究院 [3]同济大学医学院 [4]苏州瑞奇生物医药科技有限公司 [5]加利福尼亚大学洛杉矶分校人类遗传系

出　　处：《中华细胞与干细胞杂志（电子版）》2014年第1期13-16,共4页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家973重大基础研究计划(2013CB967500;2011CB65102);国家自然科学基金(81070743;81170855;81001427;81270952;81070684);江苏省自然科学基金(DK2011321)

摘　　要：目的：建立胎儿视网膜色素上皮细胞（fRPE）的原代培养方法。方法分离流产胎儿RPE，并进行体外原代培养、传代，免疫荧光检测培养RPE细胞分子标志物。以β细胞增殖诱导剂（二芳基脲衍生物WS3）刺激RPE细胞增殖，并测量其生长曲线。3组细胞比较采用单因素F检验。结果源自不同胎儿的fRPE细胞在原代及体外增殖中并未表现出明显不同。在体外扩增的4代fRPE中，100﹪的细胞表现出了良好的细胞形态。免疫荧光染色证实了体外扩增的fRPE细胞可很好的表达RPE细胞标记物。WS3未见有刺激RPE细胞体外增殖的作用。培养后不同时间三组细胞差异均有统计学意义（F=119.437~234.368,P均=0.000）。结论 fRPE细胞可在非复杂的培养环境中实现体外大量增殖，这些体外增殖的fRPE细胞可以为RPE移植细胞治疗视网膜黄斑病变提供丰富的细胞来源。Objective Age-related macular degeneration （AMD） is a common eye disease, causing irreversible blindness in senior people. One of the major pathogenic mechanisms of AMD is the dysfunction and loss of the retinal pigment epithelium （RPE）. Therefore, RPE cell transplantation is considered as a promising approach to treat AMD. Human fetal RPE （fRPE） appears to be a promising cell type for RPE cell transplantation. In this study, we aim to set-up a simple fRPE culture protocol for future clinical trials. Method fRPE cells in this experiment came from donated fetal eyes after abortion. RPE layer was dissected out from the choroid plexus under dissecting microscope. RPE patches were treated with trypsin until fully disassociated. Cell suspension was seeded on Matrigel coated plastic culture plate. When primary fRPE reached confluency within 2 weeks in culture, cells were passaged, expanded, and frozen. Results No obvious differences were observed between fRPE from different donors. fRPE cells can be continuously passaged for 3-4 passages. Virtually 100﹪cells exhibited typical RPE cell morphology during in vitro expansion. Immunostaining confirmed the expression of typical RPE markers in cultured fRPE cells. Conclusion Our results indicated that fRPE can be cultured and expanded rapidly in vitro without the alteration of RPE cell character. We suggest that fRPE cells be further tested for safety and efficacy via cell transplantation in AMD animal models before clinical trial in AMD patients.

关 键 词：视网膜 上皮细胞 细胞增殖 细胞培养技术 黄斑变性 

分 类 号：R714.5[医药卫生—妇产科学] R329.2[医药卫生—临床医学]