人正常及骨关节炎软骨细胞体外培养的对照研究

摘要目的：研究人正常软骨细胞及骨关节炎软骨细胞的体外分离、培养及鉴定方法，对其生物学特性进行对照并评价其生物学活性。方法：取人创伤性截肢与骨关节炎全膝置换的无菌膝关节软骨，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，测细胞增殖，甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行对照研究。结果：骨关节炎软骨细胞形态似成纤维细胞，生长速度明显较正常软骨细胞慢。MTT测细胞增殖显示，第2．4、6代骨关节炎软骨细胞在相同时间点大都比同代正常软骨细胞增殖速度慢（P〈0．05）。甲苯胺蓝及Ⅱ型胶原免疫组化染色显示，骨关节炎软骨细胞染色较正常软骨细胞浅，经多次传代后基本无着色。结论：正常软骨细胞5代以内细胞生长良好，生物学特性明显，5代以后出现去分化现象。骨关节炎软骨细胞增殖慢，生物学特征退变旱，符合软骨细胞退变的表现。这为骨关节炎在软骨细胞水平的研究提供了实验基础。

Control Study of Normal and Osteoarthritis Chondrocytes from Human in Vitro

Objective： To study the methods for isolation, culture and identification of articular chondrocytes fi＇om normal human and osteoarthritis patients in vitro, compare the biological characteristics and evaluate the biological activity between normal and osteoar- thritis chondrocytes. Methods Cartilage was harvested under sterile conditions flom human traumatic knee joints and osteoarthritis knee joints in Total Knee Arthroplasty. Human articular chondrocytes were isolated by using two-step enzymatic digestion, and the cells were cultured and subcultured respectively in vitro. The cells were compared by observing cell morphology under phase-contrast microscope, drawing growth curve, measuring cell proliferation, toluidine blue staining and type H collagen immunohistochemistry staining. Results： The morphology of osteoarthritis chondrocytes was similar to fibroblasts, and the growth rate was significantly more slowly than that of normal chondrocytes. Measured cell proliferation by MTT, the proliferation rate of the 2nd, 4th and 6th osteoarthritis chondrocytes was slower than that of the same generation normal chondrocytes at the same time points （P 〈0.05）. Toluidine blue staining and type II collagen immunohistochemistry staining showed that the staining of osteoarthritis chondrocytes were lighter than that of the normal chondrocytes, and were almost no coloration after several passages. Conclusion： Within 5 generations, the normal chondrocytes grew well with obviously biological characteristics and turned to dedifferentiation after 5 generations. The osteoarthritis chondrocytes with slowly cell proliferation and early degeneration of biological characteristics were in accordance with the performance of degeneration of chondrocytes, providing experimental foundation for study of the osteoarthfitis in the cellular level.