兔软骨细胞的分离及其与Ⅰ/Ⅱ型复合胶原膜共培养实验研究

Isolation of rabbit chondrocytes and their co-culture with type Ⅰ/Ⅱ collagen membrane

目的探讨体外分离兔软骨细胞的方法并观察其与Ⅰ/Ⅱ型复合胶原膜共培养时的生物活性。方法取4周龄的新西兰大耳兔,处死后在无菌条件下取关节软骨。通过胰蛋白酶和Ⅱ型胶原酶消化软骨细胞。倒置相差显微镜下观察软骨细胞特点,制作细胞爬片行甲苯胺蓝染色。将第3代兔软骨细胞与Ⅰ/Ⅱ型复合胶原膜共培养,噻唑蓝(MTT)法和糖胺聚糖含量测定法检测软骨细胞在胶原膜上的细胞活性,扫描电镜观察软骨细胞在复合胶原膜上的生长情况。结果分离出的原代软骨细胞初为卵圆形,贴壁后变成三角形或多边形,可被甲苯胺蓝染色。软骨细胞在Ⅰ/Ⅱ型复合胶原膜上生长良好。结论两部酶法可获得大量软骨细胞,MTT法和扫描电镜证实软骨细胞可在复合胶原膜上正常扩增。

Objective To explore the method of isolating rabbit chondrocytes in vitro and observe their bioactivity when co-cultured with type Ⅰ/Ⅱ collagen membrane. Methods Articular cartilage was taken under aespetic conditions after the New Zealand rabbits at 4 weeks of age were sacrificed. Chondrocytes were digested by trypsin and type Ⅱ collagenase. The features of the chondrocytes were observed under inverted phase contrast microscope. The slides of cells were stained by toluidine blue. The third-generation rabbit chondrocytes were co-cultured with type Ⅰ/Ⅱ collagen membrane. The growth of the chondrocytes on typeⅠ/Ⅱ collagen membrane was observed by the method of MTT, test of glycosaminoglycan and scaning electron microscopy. Results The original-generation chondrocytes were oval in the beginning, then they were triangle or polygan. The chondrocytes could be stained by toluidine blue. They grew well on the type Ⅰ/Ⅱ collagen membrane. Conclusions A large number of chondrocytes can be obtained by two-enzyme method. Chondrocytes can amplify normally on the type Ⅰ/Ⅱ collagen membrane, which can be demonstrated by MTT method and scaning electron microscopy.