永生化软骨细胞体外快速扩增的实验研究

In vitro amplification of immortalized chondrocytes with micro-carriers in RCCS

目的 探讨获取大量软骨组织工程种子细胞的方法和技术。方法 通过真核表达载体 ,把人端粒酶逆转录酶基因 (hTERT)转入兔髁状突软骨细胞 ,筛选、挑选阳性克隆在微载体 RCCS内快速扩增永生化软骨细胞。观测RCCS中微载体培养永生化软骨细胞的生长情况和检测细胞代谢率 ,进行Ⅱ型胶原免疫组化染色 ,并与常规培养方法进行比较。结果 永生化软骨细胞在微载体 RCCS中生长迅速、细胞代谢率高、倍增时间缩短 (P <0 0 1)。在培养第 8天 ,细胞数量可达最初接种的 95倍 (P <0 0 1)。结论 联合应用微载体和RCCS培养技术 ,能够在体外快速。

Objective To explore the method and technique to gain adequate seed cells for cartilage tissue engineering Methods hTERT gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector Rapidly proliferated immortalized chondrocytes in positive clones in micro carrier rotary cell culture system (RCCS) were screened and selected The growth of immortalized chondrocytes and the metabolic rate were observed Collagen type Ⅱ expression of immortalized chondrocytes of the experimental groups was observed The immunohistochemical results were compared with those of the control groups Results The immortalized chondrocytes in experimental groups could grow rapidly with a high metabolic rate and shorter population doubling time (PD) ( P <0 01) The amount of immortalized chondrocytes reached 95 times of the initial amount after culture for 8 d ( P <0 01) Immunohistochemistry showed that collagen type Ⅱ was positively expressed in both experimental and control groups Conclusion The immortalized chondrocytes can proliferate rapidly in large amount with the combined application of microcarrier and RCCS culture system