摘要
目的探讨重型α-地中海贫血胎儿羊水源诱导多能干细胞的建立及鉴定。方法对胎儿被诊断为重型α-地中海贫血(--SEA/--SEA)的孕妇(16+周)进行超声引导下羊膜腔穿刺术,抽取10mL羊水。利用非整合型仙台病毒介导Sox2、Klf4、c-Myc、Oct3/4四种转录因子将重型α-地中海贫血胎儿的羊水细胞重编程为诱导性多能干细胞(iPSCs);通过碱性磷酸酶(ALP)染色,免疫荧光标记(IF)检测等对iPSCs进行多能性鉴定;通过拟胚体(EB)的形成和自发分化实验对iPSCs在体内外3个胚层细胞分化的能力进行鉴定;同时进行细胞核型分析及缺失型α-地中海贫血基因诊断。结果仙台病毒可以成功地将重型α-地中海贫血胎儿的羊水细胞重新编程为非整合型诱导性多能干细胞(h-AF-SeV-iPSCs);ALP染色呈强阳性;IF检测显示Sox2、Oct4、SSEA-4、Tra-1-81表面特异性蛋白表达为阳性;h-AF-SeV-iPSCs悬浮培养均能形成囊性EB,贴壁培养后3个胚层均能分化;h-AF-SeV-iPSCs第10代及第20代细胞染色体核型均为正常核型(46,XY);且h-AF-SeV-iPSCs及其原代羊水细胞的重型α-地贫基因检测均为--SEA/--SEA缺失型。结论h-AF-SeV-iPSCs可作为研究重型α-地中海贫血胎儿宫内治疗的理想细胞模型,羊水细胞可作为iPSCs的理想细胞来源。
Objective To explore the establishment and identification of induced pluripotent stem cells sourced fetal amniotic fluid cells of a fetus with severe α-halassemia. Methods Ultrasound-guided amniocentesis was performed on pregnant women (16+ weeks) whose fetus was diagnosed with severe α-halassemia(--SEA/--SEA), 10 mL of amniotic fluid was extracted. The amniotic fluid cells of a fetus with severe α-halassemia which were reprogrammed into induced pluripotent stem cells (iPSCs) by non-integrated sendai virus mediated transcription factors of Sox2, Klf4, c-Myc, Oct 3/4. IPSCs were multifunctionally identified by alkaline phosphatase (ALP) test and immunofluorescence (IF) labeling. The ability of iPSCs to differentiate outward from three endodermal cells in vivo was identified by embryoid (EB) formation and spontaneous differentiation test. Cell karyotype analysis and genetic diagnosis of type α-thalassemia were performed. Results Sendai virus could successfully reprogram the amniotic fluid cells of a fetus with severe α-halassemia into non-integrated induced pluripotent stem cells (h-AF-SeV-iPSCs). H-AF-SeV-iPSCs were strongly positive of ALP staining, and embryonic stem cells specific proteins of SOX2, Oct4, SSEA-4 and Tra-1-81 were positive expressed of IF staining. After the suspension culture of h-AF-SeV-iPSCs, the cystic EB body could be formed, and after the adherent culture, it could also differentiate into the three germ layers. The karyotype analysis of chromosome in the 10th and 20th generation of h-AF-SeV-iPSCs were normal (46, XY). H-AF-SeV-iPSCs and its primary amniotic fluid cells were all detected as --SEA/--SEA. Conclusion The h-AF-SeV-iPSCs could be used as an ideal cell model for study of intrauterine treatment of a fetus with severe α-halassemia, and amniotic fluid cells could be used as an ideal cells source for iPSCs.
作者
张娟
赖春田
王海燕
邹闻达
彭娟
朱力宇
李辉
ZHANG Juan;LAI Chuntian;WAND Haiyan;ZOU Wenda;PENG Juan;ZHU Liyu;LI Hui(Department of Reproductive Center,Zhuzhou Central Hospital,Hu′nan Province,Zhuzhou 412007,China)
出处
《中国医药导报》
CAS
2019年第27期18-23,F0003,共7页
China Medical Herald
基金
湖南省科技计划项目(2014FJ6005)
