摘要
目的观察离心管诱导培养条件下，兔骨髓间充质干细胞（MSCs）的成软骨分化，从而为应用该技术提供实验依据。方法分离扩增兔骨髓MSCs和关节软骨细胞，采用离心管内聚集培养技术诱导培养MSCs，用含转化生长因子-β1的DMEM培养液换液，以相同培养条件下的软骨细胞为阳性对照组，以常规培养液换液的MSCs为阴性对照组；分别于培养1、2、3、4周后，收集培养物行苏木素-伊红（HE）染色、Ⅱ型胶原免疫组织化学染色和图像分析。结果MSCs诱导培养1周后开始表达Ⅱ型胶原，随时间延长而表达增强，并逐渐产生细胞外基质，但4周内表达强度始终弱于软骨细胞组（P〈0．01）。阴性对照组中部分MSCs死亡，培养物崩解。结论采用离心管诱导培养技术可以促进MSCs向软骨细胞表型分化；该技术方法操作简单、诱导确切，适宜于鉴定干细胞的软骨分化潜能。
Objective To investigate the potential of application of a culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells （MSCs）. Methods Cells obtained in bone marrow aspirates were isolated by monolayer culture from 4 rabbits and transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium inclusion TGF-β1. Chondrocyte formed the aggregates in the same medium as positive control and MSCs formed the aggregates in the conventional culture medium as negative control. Duplicate aggregates were harvested at 1 st ,2nd, 3rd and 4th week after chondrogenic differentiation to analysis. For histological evaluation,sections were stained with haematoxylin-eosin. For immunohistochemistry, sections were incubated with the monoclonal antibodies specific to type Ⅱ collagen. Results As early as first week after beginning three-dimensional culture,type Ⅱ collagen was detected in the aggregated MSCs and chondrocyte. The aggregate cultures caused an increase in the measured type Ⅱ collagen expression in two groups at different time points,but the difference being significance （P 〈0.01 ）. The induction of chondrogenesis was accompanied by an increase in the extracellular matrix. However,MSCs incubated in DMEM with 10% FBS did not form a clearly identifiable aggregate at any time. Conclusion Three-dimensional aggregate culture in a chemically defined medium facilitates the chondrogenic differentiation of rabbit MSCs.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第3期316-318,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30300079）
全军“十一五”医药卫生科研基金资助项目（06G079）
