基础医学与临床 ›› 2014, Vol. 34 ›› Issue (1): 82-87.

• 研究论文 • 上一篇    下一篇

共培养人脐带间充质干细胞(hUC-MSCs)和兔关节软骨细胞诱导hUC-MSCs分化成软骨细胞

罗二梅,张家文,胡笑珂,唐明乔,宇丽   

  1. 暨南大学
  • 收稿日期:2013-03-08 修回日期:2013-05-20 出版日期:2014-01-05 发布日期:2013-12-26
  • 通讯作者: 宇丽 E-mail:doctoryuli@yahoo.com.cn
  • 基金资助:
    广东省自然科学基金

Co-culture of Rabbit chondrocytes and Human umbilical cord mesenchymal stem cells inducse Human umbilical cord mesenchymal stem cells to differentiate into chondrocytes

  • Received:2013-03-08 Revised:2013-05-20 Online:2014-01-05 Published:2013-12-26

摘要: 目的 探讨兔膝关节软骨细胞和人脐带间充质干细胞(hUC-MSCs)的共培养对hUC-MSCs成软骨诱导分化的影响及共培养的最佳比例。方法 分离培养hUC-MSCs和兔膝关节软骨细胞并鉴定其特异性。在Transwell体系中,按1:4,1:3,1:2,1:1,2:1,3:1及4:1 的比例(hUC-MSCs:兔膝关节软骨细胞)共培养。倒置相差显微镜观察细胞的形态与增殖;甲苯胺蓝染色及免疫荧光染色分别检测葡萄糖胺聚糖(GAG)和Ⅱ型胶原(COL2-A1)(蛋白水平定性);并对各组细胞爬片进行GAG、COL2-A1定量检测;同时用实时定量荧光PCR(pPCR)检测GAG、COL2-A1mRNA表达,观察共培养前后细胞的基质分泌情况。结果 共培养 21d 后,阳性对照组和实验组细胞甲苯胺蓝染色及免疫荧光反应均呈阳性; GAG、COL2-A1含量及mRNA表达量1:4实验组均要高于其他实验组和阳性对照组。结论hUC-MSCs和兔关节软骨细胞的共培养可明显促进hUC-MSCs向软骨样细胞诱导分化,且最佳共培养比例为1:4。

关键词: Transwell小室, 人脐带间充质干细胞, 兔膝关节软骨细胞, 共培养, 成软骨诱导

Abstract: Objective To determine if the co-culture of rabbit articular chondrocytes and hUC-MSCs in vitro can affect differentiation of hUC-MSCs into cartilage-like cells, especially chondrocytes, and if so, what the optimal ratio of the two cell types is. Methods To Co-culture rabbit articular chondrocytes and hUC-MSCs at a chondrocyte: hUC-MSCs ratio of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4 for 21 days and cultured in DMEM high glucose medium. Type Ⅱcollagen (COL2-A1) and glycosaminoglycan (GAG) were analyzed qualitatively by toluidine blue and immunofluorescence technique, respectively. The contents of COL2-A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2-A1 were assayed by real-time fluorescence quantitative PCR. Results The expression of COL2-A1 and GAG on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in other the experimental group or the induced hUC-MSCs group. Also on day 21, the expression of COL2-A1 and GAG proteins in the 4:1 group was much higher than that in all other groups. Conclusion The optimal cell ratio in Transwell co-culture system appears to be 1:4 (hUC-MSCs :chondrocytes).

Key words: Transwell co-culture system, Human umbilical cord mesenchymal stem cells, Rabbit knee articular chondrocytes, Co-culture, chondrogenic induction

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