人关节软骨细胞培养法的改良及其特有表型的检测  

The modified chondrocyte culture method and inspection of cell' s phenotype

作　　者：宋卫东[1] 刘尚礼[1] 李卫平[1] 沈慧勇[1] 黄东生[1] 黄建荣[1] 

机构地区：[1]中山大学附属第二医院骨科,广州510120

出　　处：《中华实验外科杂志》2003年第2期147-148,F003,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学青年基金资助项目(30100188);广东省重点攻关项目(ZKM04903S);广东省卫生厅基金资助项目(A2000187)

摘　　要：目的探讨简便、易行的人关节软骨细胞体外培养方法。方法 以0.25％胰酶消化软骨块0.5 h,0.30％胶原酶消化4 h后,将软骨细胞与小片软骨(0.2 mm3大小)共同置入预涂多聚赖氨酸的培养瓶中培养;与传统方法对照,用倒置显微镜、免疫组织化学、电镜、逆转录-聚合酶链反应(RT-PCR)等方法,检测经7次传代后软骨细胞表型改变相伴随的形态学及分子生物学特征。结果改良法培养的细胞活性率可达100％。经多次传代的软骨细胞仍保持原代软骨细胞的表型特征,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色阳性,并可观察到软骨的特有的细胞-胶原纤维几何构像。而传统法培养的软骨细胞在第7代特有表型出现改变。结论本改良法是一种较方便而且有效的培养法。Objective To explore a simple and convenient method for human articular chondro-cyte culture and inspect cell's phenotype in vitro. Methods The chondrocytes were obtained by incomplete enzyme digestion of cartilage and were seeded with small piece of cartilage in 25 ml culture flasks coated with poly-lysine previously. After 7 passages, Toluidine blue dine was used to determine the gly-coamine and immunohistochemical method to the collagen II , phase-contrast microscopy to examine the growth, RT-PCR to detect the mRNA of precollagen II and electron microscopy to observe the ultrami-croanatomy. Results The percentage of viable chondrocytes cultured by modified method with trypan blue dine was 100% , Chondrocyte's phenotype still existed after 7 passages. The characteristic geometry of cell-collagen fibrous could be seen by microscopy. Conclusion The modified method is simple and convenient.

关 键 词：关节 软骨细胞 培养 表型 检测 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]