地中海贫血“integration-free”诱导多能干细胞的建立及造血分化的研究  

Generation of thalassemia-specific integration-free induced pluripotent stem cells and determination of their differentiation ability

作　　者：蒋满波[1,2] 曾敏慧[1] 章钧[3] 文艳飞[1] 张滨[2] 蔡柳洪[1] 

机构地区：[1]中山大学附属第三医院生殖医学中心,广东广州510630 [2]中山大学附属第三医院不育与性医学科,广东广州510630 [3]中山大学附属第三医院产前诊断实验室,广东广州510630

出　　处：《中国病理生理杂志》2015年第2期245-249,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金面上资助项目(No.81170533)

摘　　要：目的:建立无外源基因整合(integration-free)的地中海贫血患者特异诱导多能干细胞(induced pluripotent stem cells,i PSC),并研究其向造血前体细胞分化的可能性。方法:用核转染的方法将表达Oct4、Sox2、Klf4、c-Myc和Lin28转录因子的质粒p EB-C5以及表达SV40大T抗原的质粒p EB-Tg导入引产巴氏水肿胎皮肤成纤维细胞中,将其重编程为i PSC后检测其向3个胚层细胞分化的特性。将i PSC细胞与小鼠OP9细胞共培养向造血前体细胞诱导分化,检测造血前体细胞特异性抗原。结果:成功建立了巴氏水肿胎皮肤成纤维细胞来源的α地中海贫血患者特异i PSC系,其具有向3个胚层分化的能力,与OP9细胞共培养9 d后可检测到造血前体细胞标记CD34的阳性率为18.7%,CD34和CD45双阳性为12.2%。结论:巴氏水肿胎皮肤成纤维细胞可成功诱导成"integration-free"i PSC,该细胞系具有向3个胚层分化的能力,与OP9共培养可向造血前体细胞分化。AIM： To generate thalassemia-specific integration-free induced pluripotent stem cells（ i PSC） and to detect their ability of differentiation into hematopoietic precursors. METHODS： The plasmids p EB-C5 and p EB-Tg were transfected into the fibroblast cells from hemoglobin Bart＇s hydrops fetalis＇ s skin by the method of nuclear transfection to reprogramm the cells into i PSC. The ability of the i PSC to differentiate into 3-germ layer cells was determined. The i PSC were cocultured with mouse OP9 cells to differentiate into hematopoietic precursors and the hematopoietic precursor specific antigens were detected. RESULTS： The integration-free i PSC from hemoglobin Bart＇s hydrops fetalis＇ s skin fibroblasts were successfully derived,and had the ability to differentiate into 3 germ layers. When cocultured with OP9 cells for 9 d,the positive rate of hematopoietic progenitor cell marker CD34 was 18. 7%,and the CD34 and CD45 double positive rate was 12. 2%. CONCLUSION： Hemoglobin Bart＇s hydrops fetalis＇ s skin fibroblasts can be successfully induced into＂integration-free＂i PSC. This cell line has the ability to differentiate into 3 germ layers,and can be differentiated into hematopoietic precursors when cocultured with OP9 cells.

关 键 词：地中海贫血 诱导多能干细胞 无外源基因整合 造血分化 

分 类 号：R363[医药卫生—病理学] Q813.5[医药卫生—基础医学]