中文摘要：

目的 建立永生化大鼠前软骨干细胞（PSCs）株，为研究前软骨干细胞的分化机制及临床应用奠定基础。方法 用Lipofectamine^TM2000介导基因转染，将含有SV40T抗原基因的真核表达载体pCMVSV40T／PUR导入经免疫磁珠分选出的原代PSCs进行稳定表达，用嘌呤霉素筛选出阳性克隆并扩大培养，观察细胞形态及生长状况，绘制细胞生长曲线，用免疫细胞化学方法和RTopCR鉴定SV40T抗原基因在转染细胞中的表达。结果 分离获得转化细胞阳性克隆，用免疫组化证实FGFR-3表达阳性，提取RNA后用RT-PCR法成功扩增出588bp的片段。转染细胞经扩大培养，命名为永生化前软骨干细胞。贴壁培养的转染细胞群体倍增时间为（22．98±2．77）h，传代、冻存和复苏对细胞形态及生长无明显影响。结论 在体外培养条件下，可以从新生大鼠干骺端分离、培养出前软骨干细胞，pCMVSV40T／PUR转染能使其永生化。

英文摘要：

Objective To establish immortalized precartilaginous stem cells（PSCs） from neonatal SD rats in vitro for the further related research on the differentiation mechanism and clinic application of precartilaginous stem cells. Methods By using LipofectarnineTM2000, a gene transfection reagent, plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene（SV40Tag） was transfected into the primary cultured PSCs isolated by immuniomagnetic beads coasted with the second antibody. Colonies were isolated by puromycin selection and expanded by many passages. Investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by immunocytochemistry method and RTPCR. Results A particular anti -puromycin cell clone was acquired, which was confirmed as fibroblast growth factor receptor-3 （ FGFR-3 ） positive PSCs. The total RNA were isolated from the positive cell clones, and a 588 bp fragment, which was specific for the SV40T antigene gene, was amplified. The transfected cells were expanded to immortalized cell strain, named as immortalized precartilaginous stem cells （ IPSCs ）. The population doubling time of IPSCs was （ 22.98 ± 2.77 ） h, no significant effect of subculture, freezing and recovering had been found. Conclusion Precartilaginous stem cells could be isolated from neonatal SD rats, cultured in vitro, and immortalized through the transfection of pCMVSV40T/PUR.