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(COL2A1)(蛋白水平定性);并对各组细胞爬片进行GAG、COL2A1定量检测;同时用实时定量荧光PCR(pPCR)检测GAG、COL2A1 mRNA表达,观察共培养前后细胞的基质分泌情况。结果共培养21 d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、COL2A1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。
Objective To determine if the co-culture of rabbit articular chondrocytes and hUC-MSCs in vitro can af-fect 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 Ⅱ collagen（COL2A1）and glycosaminoglycan（GAG）were analyzed qualitatively by toluidine blue and immunofluores-cence technique,respectively. The contents of COL2A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2A1 were assayed by real-time fluorescence quantitative PCR. Results The expression of COL2A1 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 COL2A1 and GAG proteins in the 4∶ 1 group was much higher than that in all other groups. Conclusions The optimal cell ratio in Transwell co-culture system appears to be 1∶ 4（hUC-MSCs：chondrocytes）.
出处
《基础医学与临床》
CSCD
北大核心
2014年第1期82-87,共6页
Basic and Clinical Medicine
基金
广东省自然科学基金(9151008901000050)
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
transwell co-culture system
human umbilical cord mesenchymal stem cells
rabbit knee articular chondrocytes
co-cul-ture
chondrogenic induction
