地中海贫血患者来源外周血及脐带血细胞诱导式多能干细胞的建系和造血分化  

Establishment of the thalassemia patient-specific induced pluripotent stem cells from peripheral and cord blood cells and its hematopoietic differentiation

作　　者：曾敏慧[1] 蒋满波[2] 文艳飞[1] 彭澄[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,广州510630 [2]中山大学附属第三医院不育与性医学科,广州510630

出　　处：《广东医学》2015年第11期1633-1637,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81170533)

摘　　要：目的探讨地中海贫血(地贫)患者特异诱导式多能干细胞系(i PSCs)细胞模型的建立及造血分化能力。方法采集Hb H病引产胎儿(α地贫)的脐带血、双重杂合子β地贫输血患者(基因型为IVS-Ⅱ-654/CD17)外周血各1例,分离单核细胞后进行短期培养,核转染再程序化为i PSCs,检测i PSCs未分化状态和多能性标志物。i PSCs与OP9细胞共培养向造血干细胞诱导分化,进行吉姆萨染色检查和流式细胞术检测造血干细胞的分化结果。结果地贫患者i PSCs表达干细胞多能性标志物Oct4、SSEA4、Tra-1-60、Tra-1-81,碱式磷酸酶染色阳性,悬浮培养可以形成类球形拟胚体,畸胎瘤形成实验显示地贫患者i PSCs可分化为内、中、外胚层细胞。流式细胞术检测结果显示CD34+细胞分别为8.71%(α-i PSCs分化结果)和4.46%(β-i PSCs分化结果)。结论α、β地贫患者外周血与脐带血可再程序化为i PSCs,该细胞系具有多能分化特性,与OP9共培养能分化为造血干细胞。Objective To investigate the construction and hematopoietic differentiation of induced pluripotent stem cells （ iPSCs） from thalassemia patients .Methods Cord blood from artificial labored fetus with HbH disease （α-thalassemia） and peripheral blood from a case of heterozygote of β-thalassemia （genotype IVS-II-654/CD17） were obtained.The iPSCs were re-programed from mononuclear cells separated from obtained cord blood and peripheral blood . Hematopoietic stem cell differentiation was induced by OP 9 cell co-culture.The pluripotent markers of iPSCs were detec-ted by immunofluorescence and alkaline phosphatase staining , while embryoid bodies （ EBs） and teratoma forming experi-ments were used to detect the pluripotency .Results Pluripotent biomarkers , including Oct4, SSEA4, tra-1-60 and tra-1-81, and alkaline phosphatase , were positive on iPSCs.EBs were formed by suspended culture .Three germ layer cells were found in the teratoma test .The results of flow cytometry showed that CD 34＋cells was 4.46%and 8.71%from cord blood and peripheral sources , respectively, after co-cultured with OP9 cells.Conclusion Cord blood and periph-eral blood MNCs of patients with αorβthalassemia, can be reprogrammed into iPSCs , and differentiated into hematopoi-etic stem cells.

关 键 词：地中海贫血 诱导式多能干细胞 造血分化 

分 类 号：R733.71[医药卫生—肿瘤]