大鼠胫骨生长板软骨细胞的分离与培养鉴定

目的 探讨高效生长板软骨细胞分离方法 和体外培养条件.方法 采用二步酶消化法对6只SD大鼠胫骨生长板的软骨细胞进行分离,采用含炭吸附过的胎牛血清培养基培养,按5×105个/瓶的密度接种细胞并对软骨细胞进行形态学观察和鉴定,描绘原代软骨细胞在无激素培养基中的生长曲线.结果 软骨细胞贴壁较慢,12 h后开始附壁,第8天时90%融合,互相连接成"铺路石"样结构.原代软骨细胞胞质Ⅱ型胶原免疫着色强阳性,传代后染色减弱.结论 本研究所采用的方法 能高效快速获得原代软骨细胞,原代软骨细胞最接近体内生理状态,最适合进行实验研究.

Primary culture and identification of rat tibial growth plate chondrocytes

Objective To establish the methods of isolating and cultivating chondrocytes from rat growth plate. Methods Through two-step enzymatic digestion ( digested by 0. 25% trypsin for 30 rain, and then treated with 0. 15% collagenase il for 3 h) ,tibial growth plate chondrocytes of Sprague-Dawley rats (n =6) were isolated and the primary ehondrocytes was adjusted to a density of 5 × 105/bottle. The chondrocytes were incubated for 8-10 days in culture medium (DMEM with L-Glutamine, Hepes, without phenol red) supplemented with 10% fetal bovine serum. Collagen type Ⅱ expression was detected by histochemistry. Viability, multiplication, cell growth kinectics and morphologic changes were observed. Results Chondrocytes adhered after 12 h and formed confluent growth on day 8. Collagen type Ⅱ expression in the primary passage was positive by histochemistry, and subsequently with the extension of time, the synthesis effect of collagen Ⅱ gradually weakened. Conclusion The primary passage chondrocytes have fine biological activity and are suitable for potential experimental model for studying in vitro.