关节软骨细胞体外培养及生物学特性的实验研究

Experimental research of culture in vitro and biological characteristics of articular chondrocytes

目的:探讨关节软骨细胞培养方法及其生物学特征。方法:从4周龄新西兰乳兔关节分离静置培养软骨细胞,倒置显微镜下观察原代及传代细胞形态变化,并计数绘制生长曲线。用番红-O,甲苯胺蓝染色和免疫细胞化学法观察蛋白多糖,碱性糖胺聚糖(GAGs),Ⅱ型胶原的分泌情况,用PCR鉴定Ⅱ型胶原,Aggrecan基因的表达。结果:成功建立兔关节软骨细胞体外培养实验方法。形态学及免疫化学染色显示体外培养3代以内的软骨细胞可保持表型稳定而3代后软骨细胞呈现增殖缓慢及衰老现象。结论:本实验建立的体外培养关节软骨细胞方法简单可行,3代内的兔软骨细胞表型稳定且生物学活性较高,具备可用于实验研究的能力。

Objective： To investigate the method of culturing articular cartilage cell and its biological characteristics. Methods： Chondrocytes are separated from 4-week-old New Zealand rabbit and statically cultured. Changes of morphology of primary and pas- saged cells are observed under inverted microscope and the growth curve is counted. The synthesis level of proteoglycans,alkaline gly- cosaminoglycan （GAGs） and collagen I are acknowledged by Safranino fast green staining,toluidine blue staining and immunocyto- chemistry. And expression of collagen II and Aggrecan gene are identified by PCR. Results： The experimental method of culturing rab- bit articular chondrocytes in vitro is successfully established. It is confirmed that cultured chondrocytes in three generations can main- tain phenotypie stability by morphological and immunohistochemical staining. The dedifferentiation phenomenon is occurent after chon- droeytes of three generations. Conclusion： The established method of cultured articular cartilage cell in vitro is simple and feasible. Rabbit chondrocytes in the three generations for phenotypic stability and higher biological activity have the ability that can be used for experimental studies.