CAJ | 학술논문

目的探讨类胚体中残留未分化胚胎干细胞(ESC)的特性.方法小鼠R1和Oct-4-GFP转基因ESC细胞株,体外类胚体分化20 d后消化打散,重新给予ESC常规培养液培养.观察类胚体中残留未分化细胞形态;流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力.将残留细胞扩增后注射入裸鼠背部皮下和大腿肌肉内,6周后注射部位取材进行大体和组织学检查.结果分化20 d的类胚体中存在残留未分化ESC,生长形态呈克隆样,表达SSEA1、CD31、CD9和Oct-4等未分化ESC标志;细胞能反复传代,并可在体外再次分化和残留.残留细胞注射部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织.结论胚胎干细胞分化为类胚体后仍存在残留未分化全能ESC,残留细胞在体内、外可再次分化,并能在体外分化中再次残留.
목적 탐토류배체중잔류미분화배태간세포(ESC)적특성.방법 소서R1화Oct-4-GFP전기인ESC세포주,체외류배체분화20 d후소화타산,중신급여ESC상규배양액배양.관찰류배체중잔류미분화세포형태;류식세포의화면역형광염색검측화관찰잔류세포표면표지물급체외재차분화능력.장잔류세포확증후주사입라서배부피하화대퇴기육내,6주후주사부위취재진행대체화조직학검사.결과 분화20 d적류배체중존재잔류미분화ESC,생장형태정극륭양,표체SSEA1、CD31、CD9화Oct-4등미분화ESC표지;세포능반복전대,병가재체외재차분화화잔류.잔류세포주사부위형성기태류,류체조직중존재성숙적내배층、중배층화외배층조직.결론 배태간세포분화위류배체후잉존재잔류미분화전능ESC,잔류세포재체내、외가재차분화,병능재체외분화중재차잔류.
Objective To explore the residual undifferentiated mouse embryonic stem cells (ESCs) in embryoid bodies. Methods Mouse R1 and Oct-4-GFP transgenic ESCs were firstly cultured in suspension to form embryoid bodies (EBs). Twenty days later, EBs were digested into single cells and then re-plated in standard ESC culture condition. The morphology of residual undifferentiated cells in EBs was observed, and surface makers and in vitro redifferentiation potency of residual cells were examined by flow cytometry and immunofluoreseent staining. The residual cells were expanded and subcutaneously injected into nude mice, and the specimens were harvested from the injection site for histological analysis 6 weeks after injection. Results There were residual undifferentiated ESCs in EBs differentiated for 20 days, which displayed clonal morphology and expressed undifferentiated cell markers of ESCs, including SSEA1, CD31, CD9 and Oct-4. The cells could be differentiated to form EBs again, and could be re-expanded from secondary EBs. The residual cells were able to form teratoma at the injection site, and mature endoderm, mesoderm and ectoderm tissues could be found in teratoma tissues. Conclusion There are residual undifferentiated ESCs after differentiation of ESCs into EBs. The residual ESCs can differentiate again in vitro and in vivo, and can residue again in the in vitro differentiation.