CAJ | 학술논문

目的探讨类胚体(EBs)中残留未分化胚胎干细胞(ESCs)的数量与其致瘤性的相关性.方法小鼠R1胚胎干细胞株,体外类胚体诱导分化10d,流式细胞仪检测残留未分化ESCs表面标志SSEA-1阳性率.将第10天EBs消化打散后重新给予ESCs常规培养体系培养,观察EBs中残留未分化ESCs形态,流式细胞仪检测残留细胞表面标志物;第10天EBs消化打散后以104～2×106细胞量分别注射至裸鼠四肢肌肉内,观察不同细胞数量与畸胎瘤形成的相关性.结果 ESCs分化为EBs 10 d后有(13.5±0.75)%的细胞表达SSEA-1,提示存在残留未分化ESCs.残留未分化细胞生长形态呈克隆样,高表达SSEA-1等未分化ESCs标志.EBs消化打散后仅在注射2×l06个细胞的部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织,其余各组均未见畸胎瘤的形成.结论胚胎干细胞分化为类胚体后仍存在残留未分化胚胎干细胞,并需要一定细胞数量才具有致瘤性.
목적 탐토류배체(EBs)중잔류미분화배태간세포(ESCs)적수량여기치류성적상관성.방법 소서R1배태간세포주,체외류배체유도분화10d,류식세포의검측잔류미분화ESCs표면표지SSEA-1양성솔.장제10천EBs소화타산후중신급여ESCs상규배양체계배양,관찰EBs중잔류미분화ESCs형태,류식세포의검측잔류세포표면표지물;제10천EBs소화타산후이104～2×106세포량분별주사지라서사지기육내,관찰불동세포수량여기태류형성적상관성.결과 ESCs분화위EBs 10 d후유(13.5±0.75)%적세포표체SSEA-1,제시존재잔류미분화ESCs.잔류미분화세포생장형태정극륭양,고표체SSEA-1등미분화ESCs표지.EBs소화타산후부재주사2×l06개세포적부위형성기태류,류체조직중존재성숙적내배층、중배층화외배층조직,기여각조균미견기태류적형성.결론 배태간세포분화위류배체후잉존재잔류미분화배태간세포,병수요일정세포수량재구유치류성.
Objective To explore the correlation between the amount of residual undifferentiated embryonic stem cells (ESCs) in embryoid bodies and its tumorigenicity.Methods Mouse R1 ESCs were cultured in suspension to form embryoid bodies (EBs).Ten days later,EBs were digested into single cells and then re-plated in standard ESCs culture condition.The residual undifferentiated embryonic stem cells surface maker SSEA-1 was examined by flow cytometry in EBs.The morphology of residual undifferentiated cells in EBs were observed,meanwhile the surface marker SSEA-1 was examined by immunofluorescent staining.EBs were digested into single cells and grouped into 104,105,106,2×106,and then injected into limb muscle of nude mice.The correlation of the amount of cells and its tumorigenicity was observed.Results Residual undifferentiated ESCs were observed after EBs differentiated for 10 days,which displayed clonal morphology and expressed undifferentiated ceil markers of ESCs,such as SSEA-1.The expression rate of undifferentiated cells surface marker SSEA-1 was (13.5±0.75)％ in EBs differentiated for 10 days.Only two millions single cells harvested from EBs were able to form teratoma after being injected into muscle of nude mice for 6 weeks.Mature endoderm,mesoderm and ectoderm tissues could be found in teratoma.No teratoma formed in other groups.Conclusion A certain amount residual undifferentiated ESCs still exist after differentiation of ESCs into EBs.About 2.7× 105 undifferentiated cells are able to form teratoma by iniecting into muscle of nude mice.