长期培养人脐带间充质干细胞的生物活性及其限制性

Biological activity and restriction of human umbilical cord mesenchymal stem cells during long-term culture in vitro

背景:间充质干细胞具有多向分化能力、免疫调节能力,并可在体外进行长期培养扩增,但其体外长期培养的生物学活性变化特点及其限制性仍不十分清晰。目的:观察体外长期培养人脐带间充质干细胞的生物活性变化特点及其限制性。方法:无菌收集剖宫产新生儿脐带,经胶原酶Ⅱ消化后,用黏附生长选择法获取人脐带间充质干细胞,胰酶消化传代;取第3代细胞进行流式分析,脂肪和成骨诱导鉴定;收集不同代的细胞分别用MTT法、流式细胞仪和爬片分析法检测细胞增殖活性、细胞周期、凋亡率及黏附能力。结果与结论:人脐带间充质干细胞的细胞表型CD105+/CD29+/CD44+/CD31-/CD34-/CD45-/HLADR-,可诱导分化为脂肪细胞和成骨细胞;传代培养第3～23代细胞形态无明显变化,生长曲线基本一致,第28代后细胞增长缓慢;流式分析发现第33代细胞较第3代G0/G1减少17.9%、S+G2/M增加103.4%、细胞凋亡率增加316.7%,差异均有显著性意义(P<0.01),第3～18代细胞间G0/G1、S+G2/M、细胞凋亡率和黏附细胞数差异均无显著性意义(P>0.05)。提示在体外长期培养环境中,随着传代次数的递增,人脐带间充质干细胞的增殖及黏附能力下降,细胞凋亡率增加,总的生物活性呈逐渐下降趋势,其最佳生物活性期出现在培养第3～18代之间。

BACKGROUND：Mesenchymal stem cells（MSCs） possess multi-lineage differentiation potential and immunomodulatory effects,and can be made long-term culture in vitro.However,it is still not very clear about the restriction and biological activity feature of MSCs during long-term culture in vitro.OBJECTIVE：To investigate changes of biological activity feature and restriction of human umbilical cord MSCs（hUC-MSCs） for long-term culture in vitro.METHODS：The umbilical cord from cesarean section at term newborns was collected in sterile condition.MSCs were isolated from human umbilical cord by collagenase Ⅱ digestion.The hUC-MSCs were obtained by plastic surface adhesion selection,continuous passage culture by trypsin digestion.The cells surface antigens were identified by flow cytometry,adipogenic and osteogenic ability were determined by specially inducing culture from third generation.A series of different generation cells were collected for following analysis.The cell growth curve was analyzed by MTT assay.The cell cycle and apoptosis rate were measured by flow cytometry.The cell adhesive ability was detected by piece of climbing.RESULTS AND CONCLUSION：The cultured hUC-MSCs phenotype was CD105＋/CD29＋/CD44＋/CD31-/CD34-/CD45-/HLADR-,and could were induced toward osteoblast and adipocyte differentiation under appropriate experimental conditions.There was no significant change in the cellular morphology and the cell growth curve of subculture 3-23 generation.After subculture 28 generation,cell growth became slowly.When subculture 33 generation was compared with subculture 3 generation,the cellular number of G0/G1 phase was decrease 17.9%,and S＋G2/M phase and the cell apoptotic rate were increase 103.4% and 316.7%,respectively.Their diversity was statistical significant（P 0.01）.Within subculture 3-18 generation,their diversity of the cellular number of G0/G1 and S＋G2/M phase and the cell apoptotic rate were not statistical significant（P 0.05）.Our data show that following increase of subculture frequency,hUC-MSCs proliferate and adherent capabilities of were decrease,while its apoptosis rate increased,in condition of long-term culture in vitro.It＇s all biological activity has gradually decreasing trend.The best biological activity period appeared in 3 to 18 generations.