血红蛋白Bart′s水肿胎儿综合征诱导多能干细胞系的建立

Establishment of HB Bart′ s syndrome induced pluripotent stem cell lines

目的建立血红蛋白Bart′s水肿胎儿综合征诱导多能干细胞模型。方法通过收集血红蛋白Bart′s水肿胎儿综合征孕妇患者羊水标本,分离培养其中的羊水细胞;然后通过使用仙台病毒将h Oct3/4、h Sox2、h Klf4和h L-Myc四种因子转导羊水细胞进行重编程;最后通过形态学、碱性磷酸酶染色、干细胞多能表面标志物免疫荧光、RT-PCR、EB自发三胚层分化和畸胎瘤体内分化潜能实验对获得的诱导多能干细胞进行检测分析。结果获得的诱导多能干细胞呈现出明显的干细胞样形态,碱性磷酸酶染色、多能性标志物免疫荧光染色及逆转录PCR等检测结果均符合干细胞特征,与人胚胎干细胞特性相似。体外EB自发分化和体内畸胎瘤分化实验也证实具有完全的三胚层分化发育潜力。结论成功建立血红蛋白Bart′s水肿胎儿综合征诱导多能干细胞系,为后续疾病特异性造血分化模型的建立、疾病治疗的研究及药物开发应用奠定基础。

Objective To establish HB Bart＇s syndrome induced pluripotent stem cell model. Methods Amniotic fluid samples from pregnant women with HB Bart＇s syndrome and the amniotic fluid cells were isolated and cultured. Then, amniotic fluid ceils were transduced with h-Oct3/4, h-Sox2, h-Klf4 and h-L- Myc by using Sendai virus. Finally, The induced pluripotent stem cells were detected by morphological analysis, alkaline phosphatase staining, immunofluorescence of pluripotent markers, RT-PCR, spontaneous three germ layer differentiation by EB and differentiation potential of teratoma in vivo. Results The induced pluripotent stem cells showed obvious stem cell-like morphology. The results of alkaline phosphatase staining, pluripotency marker immunofluorescence staining and reverse transcription PCR were consistent with the characteristics of stem cells and similar to those of human embryonic stem cells. In addition, in vitro EB spontaneous differentiation and in vivo teratoma differentiation experiments also demonstrated complete development potential of the three germ layers. Conclusion The successful acquisition of induced pluripotent stem celt lines of HB Bart＇s syndrome lays the foundation for the establishment of disease-specific hematopoietic differentiation model, therapy study and the application of drugs.