人脐带血间充质干细胞分离培养的技术初探

目的：探讨人脐带血中的间充质干细胞(umibihcal cord blood mesenchymal stem cells，UBCMSCs)的分离培养方法。方法采用密度梯度离心法结合直接贴壁法分离UBCMSCs,MDEM(低糖）培养基加20%新生小牛血清培养并传代，进行观察并应用FACScan流式细胞仪检测细胞表面抗原表达情况。结果：将密度梯度离心收获的单个核细胞接种培养后，24h后即可见有少量细胞贴壁，呈圆形；48h后贴壁细胞增多，少量贴壁细施形态呈单极梭形突出；反复换液后梭形细胞明显增多，最后获得均一的贴壁细胞。培养6天后细胞开始呈对数生长。在单个核细胞中，CD44^ 细胞的比例占16．8％，CD34^ 为29．6％；经培养14天后，CD44^ 细胞显著增多，占54．4％，而CD34^ 细胞的比例降至6．0％。结论：密度梯度离心结合直接贴壁法可在体外分离培养UBCMSCs，可望用于大量制备人源性MSCs。

Exploration of Isolation and Culture of Human Umibilical Cord Blood Mesenchymal Stem Cells

Objective:To explore the isolation and culture of human umibilical cord blood mesenchymal stem cells (UBCMSCs).Methods:Human UBCMSCs were isolated from human cord blood by using density gradient centrifuging and directly adhering.The adhesive cells were purified and expanded with the medium,Expansion medium consists of DMED(LG) and 20% calf serum. The cells were observed and their immunophenotypes were determined by flow-cytometry.Results:The mononuclear cells by using density gradient centrifuging,when set in culture after 24 hours,gave rise to adherent round cells.After 48 hours the adherent cells increased and part of those exhibited single-point-shuttled.After repeated culture the uniformly shuttled cells were obtained.Within mononuclear cells,16.8% cells were all positive for CD 44 and 29.6% were all positive for CD 34.After 14-days cutured,54.4% cells were all positive for CD 44 and 6.0% were all positive for CD 34.Conclusions:Human UBCMSCs can be isolated from human cord blood by using density gradient centrifuging and directly adhering.The method is used to preparation large quantity of human MSCs.