中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
Chinese Journal of Medical Genetics
2015年
5期
609-614
,共6页
胡志青%胡旭昀%庞佳伦%王晓琳%林彭思远%李卓%吴涌%邬玲仟%梁德生
鬍誌青%鬍旭昀%龐佳倫%王曉琳%林彭思遠%李卓%吳湧%鄔玲仟%樑德生
호지청%호욱윤%방가륜%왕효림%림팽사원%리탁%오용%오령천%량덕생
血友病A%诱导多潜能干细胞%内皮细胞
血友病A%誘導多潛能榦細胞%內皮細胞
혈우병A%유도다잠능간세포%내피세포
Hemophilia A%Inducible pluripotent stem cells%Endothelial cells
目的 通过逆转录病毒方法诱导建立两例血友病A(hemophilia A,HA)患者特异性诱导多潜能干细胞(inducible pluripotent stem cells,iPSCs)并定向分化为内皮细胞,为HA的发病机制以及细胞和基因治疗研究提供理想的细胞来源.方法 收集培养血友病A患者尿液细胞,使用携带Oct4、Sox2、c-Myc和Klf4等4种因子的逆转录病毒感染尿液细胞,诱导出HA患者特异性的诱导多潜能干细胞;通过形态学、干细胞多潜能表面标志物免疫荧光检测,成畸胎瘤实验鉴定iPSCs;iPSCs与OP9细胞共培养分化为内皮细胞.结果 成功建立了HA患者特异性iPSCs,其形态与人胚胎干细胞相似,细胞表面标志物免疫荧光检测均符合干细胞基因表达特征,成畸胎瘤实验显示其可在体内随机分化为三胚层组织;将iPSCs定向分化为内皮细胞,细胞表面标志物CD144、CD31、vWF免疫荧光均为阳性.结论 尿液细胞可以重编程得到血友病A患者特异性iPSCs,并能定向分化为内皮细胞,为血友病A研究提供细胞模型,也为后续基因修正后的细胞治疗提供了自体化的细胞来源.
目的 通過逆轉錄病毒方法誘導建立兩例血友病A(hemophilia A,HA)患者特異性誘導多潛能榦細胞(inducible pluripotent stem cells,iPSCs)併定嚮分化為內皮細胞,為HA的髮病機製以及細胞和基因治療研究提供理想的細胞來源.方法 收集培養血友病A患者尿液細胞,使用攜帶Oct4、Sox2、c-Myc和Klf4等4種因子的逆轉錄病毒感染尿液細胞,誘導齣HA患者特異性的誘導多潛能榦細胞;通過形態學、榦細胞多潛能錶麵標誌物免疫熒光檢測,成畸胎瘤實驗鑒定iPSCs;iPSCs與OP9細胞共培養分化為內皮細胞.結果 成功建立瞭HA患者特異性iPSCs,其形態與人胚胎榦細胞相似,細胞錶麵標誌物免疫熒光檢測均符閤榦細胞基因錶達特徵,成畸胎瘤實驗顯示其可在體內隨機分化為三胚層組織;將iPSCs定嚮分化為內皮細胞,細胞錶麵標誌物CD144、CD31、vWF免疫熒光均為暘性.結論 尿液細胞可以重編程得到血友病A患者特異性iPSCs,併能定嚮分化為內皮細胞,為血友病A研究提供細胞模型,也為後續基因脩正後的細胞治療提供瞭自體化的細胞來源.
목적 통과역전록병독방법유도건립량례혈우병A(hemophilia A,HA)환자특이성유도다잠능간세포(inducible pluripotent stem cells,iPSCs)병정향분화위내피세포,위HA적발병궤제이급세포화기인치료연구제공이상적세포래원.방법 수집배양혈우병A환자뇨액세포,사용휴대Oct4、Sox2、c-Myc화Klf4등4충인자적역전록병독감염뇨액세포,유도출HA환자특이성적유도다잠능간세포;통과형태학、간세포다잠능표면표지물면역형광검측,성기태류실험감정iPSCs;iPSCs여OP9세포공배양분화위내피세포.결과 성공건립료HA환자특이성iPSCs,기형태여인배태간세포상사,세포표면표지물면역형광검측균부합간세포기인표체특정,성기태류실험현시기가재체내수궤분화위삼배층조직;장iPSCs정향분화위내피세포,세포표면표지물CD144、CD31、vWF면역형광균위양성.결론 뇨액세포가이중편정득도혈우병A환자특이성iPSCs,병능정향분화위내피세포,위혈우병A연구제공세포모형,야위후속기인수정후적세포치료제공료자체화적세포래원.
Objective To generate hemophilia A (HA) patient-specific inducible pluripotent stem cells (iPSCs) and induce endothelial differentiation.Methods Tubular epithelial cells were isolated and cultured from the urine of HA patients.The iPSCs were generated by forced expression of Yamanaka factors (Oct4, Sox2, c-Myc and Klf4) using retroviruses and characterized by cell morphology, pluripotent marker staining and in vivo differentiation through teratoma formation.Induced endothelial differentiation of the iPSCs was achieved with the OP9 cell co-culture method.Results Patient-specific iPSCs were generated from urine cells of the HA patients, which could be identified by cell morphology, pluripotent stem cell surface marker staining and in vivo differentiation of three germ layers.The teratoma experiment has confirmed that such cells could differentiate into endothelial cells expressing the endothelial-specific markers CD144, CD31 and vWF.Conclusion HA patient-specific iPSCs could be generated from urine cells and can differentiate into endothelial cells.This has provided a new HA disease modeling approach and may serve as an applicable autologous cell source for gene correction and cell therapy studies for HA.
