人膝关节软骨下骨成骨细胞和软骨细胞的共培养  

Co-culture of subchondral osteoblasts and chondrocytes from human knee joint

作　　者：张立智[1] 蒋垚[2] 毛彦杰[2] 黄成龙[2] 

机构地区：[1]上海市杨浦区中心医院,同济大学附属杨浦医院,上海市200090 [2]上海交通大学附属第六人民医院,上海市200233

出　　处：《中国组织工程研究》2013年第11期1909-1916,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30772182);2012年度上海市卫生局局级科题项目资助(20124303)~~

摘　　要：背景:软骨细胞移植等组织工程方法已经成为非常有潜力的骨关节炎治疗措施,软骨下骨的成骨细胞对软骨细胞的作用不仅参与骨关节炎的发生和发展,而且与细胞移植治疗预后有着密切的关系。目的:培养人膝关节软骨下骨成骨细胞和软骨细胞,构建两者共培养的细胞培养模型,探讨软骨下骨成骨细胞对软骨细胞的作用。方法:收集骨关节炎患者行全膝关节置换和严重创伤患者行截肢后遗弃的胫骨平台组织,采用Ⅰ型胶原酶连续消化后进行贴壁培养的方法,培养软骨下骨的成骨细胞,应用Ⅱ型胶原酶消化方法,培养软骨细胞,然后应用多孔插入器细胞培养池构建成骨细胞和软骨细胞三维共培养模型。结果与结论:通过免疫组化,NBT/BCIP染色,茜素红染色等检查,证实成功培养出成骨细胞和软骨细胞。实时定量RT-PCR显示,在与骨关节炎的软骨下骨成骨细胞共培养的软骨细胞中,Ⅱ型胶原和蛋白聚糖基因的表达明显下降。可见骨关节炎患者的成骨细胞能促进软骨细胞的退变。BACKGROUND： Cartilage tissue engineering provides a potential method for the treatment of osteoarthritis. The relation and mechanism of interaction that subchondral osteoblasts act on chondrocytes are worthy of further research. OBJECTIVE： To co-culture subchondral osteoblasts and chondrocytes from human knee joint, thereby constructing a co-culture system of subchondral osteoblasts and chondrocytes to investigate the reaction between osteoblasts and chondrocytes. METHODS： Subchondral bone specimens were obtained from patients undergoing total knee replacement for primary osteoarthritis and those receiving amputation surgeries for serious limb injuries. Primaryosteoblasts were isolated from subchondral bone specimens using sequential digestion method, and identified using type Ⅰ collagen immunochemistry. Primary chondrocytes were isolated from articular specimens after amputation and identified using type Ⅱ collagen immunochemistry. Then, a three-dimensional co-culture system of osteoblasts and chondrocytes was constructed using cell culture inserts. RESULTS AND CONCLUSION： Osteoblasts and chondrocytes were identified successfully using immunochemistry, NBT/BCIP staining, and alizarin red staining. Real-time quantitative reverse transcription-PCR results exhibited that expression of type Ⅱ collagen and proteoglycans significantly decreased in chondrocytes co-cultured with subchondral osteoblasts from osteoarthritis patients. These findings show that osteoblasts from osteoarthritis patients can initiate chondrocyte phenotypes shifting towards degenerative differentiation.

关 键 词：组织构建 软骨组织构建 软骨下骨 成骨细胞 软骨细胞 骨关节炎 共培养 实时定量RT-PCR Ⅱ型胶原 蛋白聚糖 Ⅰ型胶原 碱性磷酸酶 国家自然科学基金 组织构建图片文章 

分 类 号：R318[医药卫生—生物医学工程]