【摘要】 目的探讨兔关节软骨细胞分离培养的方法和生物学特性。方法从4周龄新西兰白兔关节分离培养软骨细胞,倒置显微镜下观察原代及传代培养的细胞形态学变化,并计数绘制生长曲线。测定细胞冻存复苏后的存活率。用甲苯胺蓝染色和免疫细胞化学法了解葡萄糖胺多糖(GAGs),Ⅱ型胶原的合成情况并鉴定细胞。结果成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形,传代3次后出现反分化。形态学、免疫化学染色显示细胞培养3代以内可以保持表型的稳定,冷冻复苏存活率为92%。结论本文建立的体外培养关节软骨细胞的方法简单可行。体外培养的兔软骨细胞表型能保持3代稳定,具有良好的生物学活性,可用于实验研究,可经深低温冷冻保存。更多还原

【Abstract】 Purpose To develop and evaluate the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies. Methods Articular chondrocytes were isolated from the cartilages of four week-old New Zealand rabbits. The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day. Cell number was counted and growth curve was depicted. Toluidine blue staining and immunocytochemistry were used to indentify the chondrocytes and determine the synthesis of glycosaminoglycans (GAGs) and type Ⅱ collagen. The survival rate of chondrocyte post-thawing was observed. Results The primarily cultured chondrocytes was in polygonal shape, and became dedifferentiation after 3 passage. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages and the survival rate was 92 after recovering. Conclusions The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages and could survive in the condition of deep-frozening. The growth was robust and may be suitable for most experiments.更多还原