诱导人脐带间充质干细胞分化为视网膜色素上皮样细胞的研究  

Differentiation of human umbilical cord mesenchymal stem cells into retinal pigment epithelial like cells

作　　者：李鹏[1] 李宗义[2] 王丽[3] 范氏雪幸 田海滨[1] 王方[2] 吕立夏[1] 徐国彤[1] 

机构地区：[1]同济大学医学院眼科研究所及干细胞研究中心,上海200092 [2]同济大学附属第十人民医院眼科 [3]同济大学生命科学与技术学院

出　　处：《中华细胞与干细胞杂志（电子版）》2014年第1期35-40,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家重大科学研究计划(2013CB967501);国家自然科学基金(C120111;31171419);高等学校博士学科点专项科研基金(20100072120051)

摘　　要：目的探讨用猪视网膜色素上皮细胞(RPE)条件培养基诱导hUCMSCs分化为RPE样细胞的方法。方法通过流式细胞技术鉴定hUCMSCs的表面标记分子;通过诱导hUCMSCs分化成为脂肪、骨和软骨细胞确定其多系分化能力;将hUCMSCs培养在猪RPE的条件培养液中并添加诱导因子来诱导hUCMSCs向RPE细胞分化。对照组与处理组Q-PCR结果采用t检验比较。结果 hUCMSCs表达CD105、CD90、CD73、CD44和CD29,但不表达CD34,CD45和MHCII等分子标记,在成脂、成骨、成软骨分化培养基中可分化为脂肪、骨和软骨细胞。单独使用猪RPE条件培养液不能有效诱导hUCMSCs向RPE细胞分化,但联合应用猪RPE条件培养液和诱导因子(视黄酸,activin-A和人重组骨形成蛋白-7)可有效诱导hUCMSCs分化为RPE样细胞,RPE细胞标记分子RPE65、Mitf和Ck8/18的基因表达量分别提高了2.1±0.4、6.8±1.3和2.5±0.3倍(P<0.05)。诱导产生的RPE样细胞呈多边形,但不含色素颗粒。结论猪RPE条件培养液联合诱导因子可有效诱导hUCMSCs分化为RPE样细胞,可能会为治疗视网膜变性疾病提供合适的种子细胞。Objective The functions of the conditioned medium from porcine retinal pigment epithelial cells （RPE） on differentiation of hUCMSCs into RPE cells were investigated. Methods hUCMSCs were cultured in DMEM/F12 medium plus 10﹪FBS and MSC specific markers on hUCMSCs were confirmed by flow cytometry. Porcine RPE conditioned media were obtained by culturing porcine RPE cells in DMEM/F12 plus 2 ﹪ FBS for 24 h. For differentiation, hUCMSCs were cultured in RPE conditioned medium or RPE conditioned medium plus retinoic acid （RA）, Activin-A and bone morphogenetic protein-7 （BMP-7） for two weeks. hUCMSC derived RPE-like cells were confirmed by detecting the expressions of specific markers via PCR. Results hUCMSCs were positive for CD105, CD90, CD73, CD44, CD29 and negative for CD34, CD45, MHCII. RPE conditioned medium alone could not promote hUCMSCs to differentiate into RPE cells. However, Rpe65＋Mitf＋Ck8/18＋RPE-like cells could be derived from hUCMSCs when cultured in RPE conditioned medium plus RA, Activin-A and BMP-7. RPE-like cells exhibited polygonal morphology, but pigments were not observed in these differentiated cells. Conclusion RPE conditioned medium combined with RA, Activin-A and BMP-7 can promote hUCMSCs to differentiate into RPE-like cells, which may provide enough cells for clinical treatment of patients with retinal degeneration disease.

关 键 词：脐带 间充质干细胞 视网膜 上皮细胞 视网膜变性 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]