小鼠羊水间充质干细胞的分离、培养及鉴定

Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells

目的探讨小鼠羊水间充质干细胞(AF-MSC)的分离、培养及鉴定。方法在无菌条件下获取孕鼠子宫,收集羊水后进行过滤和离心,对沉淀细胞团进行培养并传代。观察AF-MSC的形态,分析AF-MSC的增殖特点,采用流式细胞术鉴定AF-MSC的表面标志物,检测AF-MSC的三系分化能力及冷冻复苏后的细胞活力。结果小鼠AF-MSC呈典型的梭形,融合度>80%时会出现典型的漩涡状结构。小鼠AF-MSC传代培养无明显潜伏期,培养2~3 d进入对数增长期,增长速度最快,之后增殖速度减慢,进入平台期。AF-MSC表达干细胞抗原(Sca)-1、CD29、CD44,不表达CD34、CD45。小鼠AF-MSC成骨分化后,矿化结晶被茜素红染成深红色的点状;成软骨分化后,分泌的酸性粘多糖被阿利新蓝染成淡蓝色;成脂分化后,胞质脂滴被油红O染成红色。细胞冷冻复苏后存活率>95%,生长状态良好,6 d时增殖能力高于冻存前(P<0.05),其他时间增殖能力与冻存前比较,差异无统计学意义(均为P>0.05)。结论本实验成功分离了小鼠AF-MSC,过程简便、成本低,且分离的细胞可随传代次数的增加而纯化,冻存不影响其增殖能力。

Objective To explore the isolation,culture and identification of mouse amniotic fluid-derived mesenchymal stem cell(AF-MSC).Methods The uteruses of pregnant mice were obtained under sterile conditions.The amniotic fluid was collected,filtered and centrifuged,and the precipitated cell mass was cultured and passaged.The morphology of AF-MSC was observed and the proliferation characteristics of AF-MSC were analyzed.The surface markers of AF-MSC were identified by flow cytometry.The osteogenic,chondrogenic and adipogenic differentiation capability of AF-MSC and cell vitality after cryopreservation and resuscitation were evaluated.Results The mouse AFMSC was seen in typical spindle shape,and vortex structure could be observed when the cell confluency exceeded 80%.No evident latency was noted in the passage and culture of mouse AF-MSC.After 2-3 d culture,AF-MSC proliferated in the logarithmic growth stage with the fastest growth rate,which was slowed down and entered into the plateau period.AF-MSC expressed stem cell antigen(Sca)-1,CD29 and CD44 rather than CD34 and CD45.After the osteogenic differentiation of mouse AF-MSC,the mineralized crystals were stained in dark red spots by Alizarin red S staining.After chondrogenic differentiation,the secreted acid mucopolysaccharide was stained in light blue by Alcian blue.After adipogenic differentiation,cytoplasmic lipid droplets were stained in red by oil red O staining.After cryopreservation and resuscitation,the survival rate of AF-MSC exceeded 95%,and the growth status was excellent.The proliferation ability at 6 d was significantly better than that before cryopreservation(P<0.05),and the proliferation ability at other time points did not significantly differ from that before cryopreservation(all P>0.05).Conclusions Mouse AF-MSC may be successfully isolated with convenient procedure and the low cost.In addition,the isolated AF-MSC may be purified along with the increasing times of passage.Cryopreservation does not affect the proliferation ability of AF-MSC.