SV40诱导髁突软骨细胞永生化的实验研究

Immortalization of the rabbit mandibular condylar chondrocyte with SV40

目的 建立髁突软骨细胞的永生化细胞系。方法 构建含有SV40大T抗原基因和neo抗性基因的逆转录病毒载体pLN/SV40 ,用常规脂质体转染的方法将目的基因导入包装细胞PA317,G418筛选出阳性克隆后 ,收集其病毒上清感染原代培养的新西兰白兔髁突软骨细胞 (mandibularcondylarchondrocyte ,MCC) ,G418筛选阳性克隆并挑选单克隆。设计大T抗原基因的特异性引物 ,用PCR检测不同克隆株对目的基因的整合。对所挑选的阳性克隆株进行扩增培养及常规的冻存复苏 ,比较其与MCC在形态增殖等方面特征。结果 10个阳性克隆株成功扩增出了 1196bp的基因片断 ,有 5株细胞传代超过 30次 ,其中克隆株A2 1已在体外 1.5年稳定传代 10 0次以上 ,命名为永生化髁突软骨细胞 (immortalizedmandibularcondylarchondrocyte,IMCC)。IMCC和MCC的倍增时间分别是 30 86h和 78 95h。IMCC的形态以多角形为主 ,传代、冻存和复苏对细胞形态及生长无明显影响。结论 已建立了 1株永生化的髁突软骨细胞系。

Objective To establish an immortalized cell line of the mandibular condylar chondrocyte (MCC). Methods Reconstructed retroviral vector pLN/SV40 containing the large T antigen and neomycin resistance genes were transfected into the packaging cells PA317 using lipofectin transfection method. Then the primarily cultured cells derived from mandibular condylar chondrocytes(MCCs) of New Zealand rabbits were infected with the positive retrovirus medium and monoclone was selected with G418. Specific primer of large T antigen was designed and PCR was used to detect the integrating of large T antigen gene. The positive cell clones were subcultrued and their growth characteristics were compared with untransfected cells. Results 10 cell clones of the transduced cells showed the positive gene fragment of 1 196 bp, and 5 of them had been further subcultured for more than 30 passages. One clone of A21 had been passaged for more than 100 times in nearly one and half year, and named immortalized mandibular condylar chondrocyte (IMCC). IMCCs were polygonal shaped, similar to MCCs; the population doubling time of IMCC and MCC were 30 86 h and 78 95 h respectively. Subculture, freezing and recovering had no effect on cellular shape and proliferation of IMCC. Conclusions An immortalized cell cline derived from the rabbit MCC has been successfully established.