CAJ | 학술논문

目的:观察人脐带间充质干细胞(human umbiiicai cord derived mesenchymai stem ceiis ,hUCMSCs)体外长期传代扩增后形态表型、增殖及分化特性的变化. 方法:分别以双酶消化及组织块法分离培养hUCMSCs;细胞传至18代(P18),观察细胞形态、生长曲线、克隆形成、细胞周期、干细胞表型、多向分化能力的变化,全面评测连续传代对hUCMSCs生物学特性的影响.结果:双酶消化结合组织块法可高效分离获得大量hUCMSCs ,细胞稳定传代并保持间充质干细胞特性;早期细胞多呈长梭形或纺锤形,至P18时呈多角不定型且细胞体积变大,胞浆增加明显. 传代至P18后,hUCMSCs的群体倍增时间增加至60 h以上,绝大多数细胞处于G0/G1期,且成纤维细胞集落形成单位( coiony-forming unit-fibrobiast,CFU-F)形成降低,克隆集落减小.hUCMSCs可表达且不因持续传代丢失间充质干细胞标记,但仅早期传代细胞表达多能干细胞标记Oct-4及SSEA-4. 特异性染色及相关基因表达检测提示P18 hUCMSCs的成脂肪、成软骨及成骨诱导分化能力较早期传代细胞减弱. 结论:hUCMSCs是具备高增殖活性和多向分化特性的间充质干细胞群,体外长期传代扩增的hUCMSCs呈现一定的复制性衰老趋势,但不会丢失干性,可稳定表达间充质干细胞特异性表面标记并保持分化活性.
목적:관찰인제대간충질간세포(human umbiiicai cord derived mesenchymai stem ceiis ,hUCMSCs)체외장기전대확증후형태표형、증식급분화특성적변화. 방법:분별이쌍매소화급조직괴법분리배양hUCMSCs;세포전지18대(P18),관찰세포형태、생장곡선、극륭형성、세포주기、간세포표형、다향분화능력적변화,전면평측련속전대대hUCMSCs생물학특성적영향.결과:쌍매소화결합조직괴법가고효분리획득대량hUCMSCs ,세포은정전대병보지간충질간세포특성;조기세포다정장사형혹방추형,지P18시정다각불정형차세포체적변대,포장증가명현. 전대지P18후,hUCMSCs적군체배증시간증가지60 h이상,절대다수세포처우G0/G1기,차성섬유세포집락형성단위( coiony-forming unit-fibrobiast,CFU-F)형성강저,극륭집락감소.hUCMSCs가표체차불인지속전대주실간충질간세포표기,단부조기전대세포표체다능간세포표기Oct-4급SSEA-4. 특이성염색급상관기인표체검측제시P18 hUCMSCs적성지방、성연골급성골유도분화능력교조기전대세포감약. 결론:hUCMSCs시구비고증식활성화다향분화특성적간충질간세포군,체외장기전대확증적hUCMSCs정현일정적복제성쇠로추세,단불회주실간성,가은정표체간충질간세포특이성표면표기병보지분화활성.
Objective:To investigate the effect of in vitro extended passaging on the phenotype ,proiiferation and differentiation char-acteristics of human umbiiicai cord mesenchymai stem ceiis (hUCMSCs).Methods:Methods of enzymatic digestion,tissue expiantation and combination of the two were compared for isoiation of hUCMSCs .Ceii morphoiogy,growth curve,CFU-F forming abiiity,ceii cycie, phenotype and differentiation activity of hUCMSCs after expanding over eighteen passages in vitro were comprehensiveiy assessed .Re-sults:Combination of enzymatic digestion and tissue expiantation was the best way to successfuiiy isoiate abundant hUCMSCs .After iong time expansion in vitro,morphoiogy of hUCMSCs changed from eiongated and spindied -shape with narrow cytopiasm to fiattened-shape with more iameiiopoid .Popuiation doubiing time ( PDT) increased to more than 60 hours and CFU-F forming abiiity significantiy de-creased at P18 suggesting the repiicative senescence of hUCMSCs .Mitotic frequency showed more ceiis in G 0/G1-phase,indicating the existence of iower percentage of proiiferating ceiis fraction after iong time subsequent passages .MSCs marhers were consistentiy ex-pressed during passaging ,whiie piuripotent stem ceiis marhers Oct-4 and SSEA-4 were negativeiy expressed at iate passages .Specific staining and gene expression detection showed attenuated differentiation viabiiity of hUCMSCs at P 18.Conclusions:HUCMSCs were MSCs subpopuiation with high proiiferation and differentiation viabiiity .Aithough ceiiuiar senescence of hUCMSCs was noted after iarge amount of expansion in vitro,they stiii possess stabie expression of MSCs marhers and the abiiity of muiti-differentiation.