猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的构建

Construction of immortalized rat astrocyte strain by transfection of simian virus 40 large T antigen gene

目的 构建永生化大鼠星形胶质细胞,为转基因细胞移植镇痛提供细胞载体。方法采用差速粘附法体外分离和培养大鼠大脑皮层星形胶质细胞。利用脂质体将含有猿肾病毒40大T抗原(SV40Tag)基因的质粒pCMVSV40T／PUR转染培养的大鼠星形胶质细胞。经嘌呤霉素1.5μg/ml筛选后,挑选阳性细胞克隆扩大培养并连续传代。PCR、RT-PCR及免疫组化检测阳性细胞克隆中SV40Tag基因的整合情况及其表达,并对传代细胞的胶质原纤维酸性蛋白(GFAP)进行检测。结果成功获得体外培养的大鼠星形胶质细胞,GFAP阳性表达;筛选获得阳性细胞克隆,连续传代培养近50代;PCR、RT-PCR产物经1.5％琼脂糖凝胶电泳分析显示558bp处有一特异性扩增条带,与阳性对照条带相同,而未转染pCMVSV40T／PUR的细胞无扩增条带,回收片段经测序、比对与SV40Tag的基因序列一致(100％);同时转染的阳性细胞克隆SV40Tag和GFAP免疫染色阳性。结论 成功地构建了SV40Tag基因永生化的大鼠星形胶质细胞株。

Objective To immortalize rat astrocytes which could be used as cell carriers for transgenic cellular analgesia. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats by trypsin digestion and cultured according to the method of differential cell adhesiveness and transfected with plasmid pCMVSV40T/ PUR containing the simian virus 40 large tumor antigen (SV40Tag) gene. The positive colonies were isolated by puromycin selection and expanded by many passages. The integration and expression of large T antigen gene were detected and the immuno-reactivity of glial fibrillary acidic protein (GFAP) was determined by PCR, RT-PCR and immuno-cytochemistry. Results Rat astrocytes were successfully cultured in vitro and positively stained for the astrocytic marker GFAP. The positive colonies were isolated and subcultured for 50 passages. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis. The size of amplifacation product of target gene was identical to that of the positive control (558 bp) . There was no PCR and RT-PCR product from non-transfected cells. DNA sequencing and BLAST showed that 558 bp nucleotides were identical to the SV40Tag gene of the Genbank (100%). The transfected cells were positively stained for the SV40Tag and GFAP. Conclusion Immortalized rat astrocyte strain with SV40 tag gene is constructed successfully.