吴航飞, 王康淳, 潘崎, 等. 小鼠羊水间充质干细胞的分离、培养及鉴定[J]. 器官移植, 2022, 13(1): 67-73. DOI: 10.3969/j.issn.1674-7445.2022.01.011
引用本文: 吴航飞, 王康淳, 潘崎, 等. 小鼠羊水间充质干细胞的分离、培养及鉴定[J]. 器官移植, 2022, 13(1): 67-73. DOI: 10.3969/j.issn.1674-7445.2022.01.011
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Wu Hangfei, Wang Kangchun, Pan Qi, et al. Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells[J]. ORGAN TRANSPLANTATION, 2022, 13(1): 67-73. DOI: 10.3969/j.issn.1674-7445.2022.01.011
Citation: Wu Hangfei, Wang Kangchun, Pan Qi, et al. Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells[J]. ORGAN TRANSPLANTATION, 2022, 13(1): 67-73. DOI: 10.3969/j.issn.1674-7445.2022.01.011
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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

    摘要
  • 摘要:
      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。

     

    Abstract:
      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 AF-MSC 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.

     

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