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目的：研究人关节软骨细胞的体外分离、培养及鉴定方法，观察各代人关节软骨细胞的形态学特性。方法：取人创伤性截肢的无菌膝关节软骨，采用两步酶消化法分离培养人关节软骨细胞，并进行传代培养。通过倒置相差显微镜下观察细胞形态，绘制生长曲线，甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行鉴定。结果：两步酶消化法消化出的软骨细胞呈圆形，培养2-3 天，细胞贴壁、变形，呈三角形或多角形，2 周左右细胞融合成层，传代5 次后出现去分化。软骨细胞增殖和生长缓慢。形态学、免疫组织化学染色显示细胞培养5 代以内可以保持表型的稳定。结论：本研究采用胰蛋白酶及Ⅱ型胶原酶联合消化法获得大量高纯度、高活性的人关节软骨细胞。5 代以内细胞生长良好，生物学特性明显，适合于实验研究，5 代以后出现去分化现象。

英文摘要:

Objective: To study the methods for isolation, culture and identification of human articular chondrocytes in vitro, and observe the morphological characteristics of human articular chondrocytes in different generations. Methods: Cartilage was harvested under sterile conditions from human traumatic knee joints, human articular chondrocytes were isolated and cultured by using two-step enzymatic digestion, and the cells were subcultured. The cells were identified by observing cell morphology under phase-contrast microscope, drawing growth curve, toluidine blue staining and type Ⅱ collagen immunohistochemistry staining. Results: The cells were round from the two-step enzymatic digestion, cells showed adhesion, deformation, triangle or multiangle after two-three days cultured and the cells formed a cohesive multiplayer on plastic surface about two weeks, and turned to dedifferentiation after five generations. The proliferation and growth of chondrocytes was slowly. Morphology and immunohistochemistry staining showed that stable phenotype could be maintained within five generations. Conclusion: In this study, a lot of highly pure and active human articular chondrocytes were obtained by using trypsin and type Ⅱ collagenase digestion. Within five generations, the cells grew well with obviously biological characteristics, suitable for experimental study, and turned to dedifferentiation after five generations.

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