体外传代对人脐带间充质干细胞生物学特性的影响

Effect of in vitro extended passaging on the biological characterization of human umbilical cord mesenchymal stem cells

目的:观察人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,h UCMSCs)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h UCMSCs;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h UCMSCs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h UCMSCs,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,h UCMSCs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(colony-forming unit-fibroblast,CFU-F)形成降低,克隆集落减小。h UCMSCs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 h UCMSCs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:h UCMSCs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h UCMSCs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。

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.