猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的生物学特性  

Biological character of immortalized rat astrocyte strain by transferction of simian large T antigen gents

作　　者：安珂[1] 田玉科[1] 杨辉[1] 高峰[1] 王鹏[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室

出　　处：《中华麻醉学杂志》2005年第6期441-444,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目(30170905)

摘　　要：目的研究猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株(IAST)的生物学特性,探讨其作为转基因细胞镇痛载体的可行性。方法取大鼠大脑皮层星形胶质细胞(AST)和IAST 进行体外培养,观察细胞的形态学、超微结构以及传代复苏的情况;绘制细胞生长曲线,免疫细胞化学法检测细胞胶质纤维酸性蛋白的表达;5’溴-2-脱氧尿苷掺入法和流式细胞仪检测细胞增殖周期;双层软琼脂克隆形成实验和裸鼠接种实验检测细胞的致瘤性。结果AST体外传代至10代左右即出现复制衰老现象,而IAST可连续传代,未出现衰老迹象;细胞生长呈单层,锚着依赖和接触抑制;传代、冻存和复苏对IAST的存活率无影响,而第6代和第10代AST的存活率降低(P<0.01);与第6代和第10代AST相比,IAST增殖较为旺盛,增殖指数较高,群体倍增时间较短、增殖率较高(P<0.01); IAST胶质纤维酸性蛋白免疫染色阳性,软琼脂培养无集落形成,裸鼠接种无致瘤性。结论大鼠IAST是增殖活跃的非恶性转化细胞,可安全地用于转基因细胞移植镇痛研究。Objective To study the biological character of rat astrocyte strain immortalized by simian virus 40 large T antigen gene（SV40Tag） and explore the feasibility of using it as cell vehicle for transgenic cellular analgesia. Methods Rat cerebral cortical astrocytes （AST） and immortalized rat astrocyte strain （IAST） were cultured in vitro. Morphology and growth features of AST and IAST were examined and compared after subculture,freezing and recovery. The ultrastructure of the cells was observed by transmission electron microscopy. The glial fibrillary acidic protein （GFAP） in these cells was detected by immuno-cytochemistry. The cell proliferation rate and cell cycle were determined by bromodeoxyuridine labelling and flow cytometry. AST and IAST were cultured in soft agar and inoculated in nude mice to investigate the tumorigenesis of IAST. Results IAST could be subcultured successively. The cells remained monolayer, anchorage dependent and the growth was attachment-inhibited. When AST was subcultured for only 10 passages, replicative senescense began. Subculture, freezing and recovery did not influence the shape and proliferation of IAST （94% ± 5% ）, but decreased the vitality rate of AST （54% ± 4% ）（P 〈0.01） . The proliferation capability of IAST was stronger than that of AST. The proliferation index was increased （9.6% ± 2.6% vs 30.7% ± 3.4% ） （ P 〈 0.01） ; population doubling time was shortened [（50.0±3.2）hvs （19.6±2.1）h]（P〈0.01） and proliferation rate was increased （21% ±3% vs72% ±7%） （P〈0.01 ）. Moreover IAST was positively immunostained for GFAP and retained its original differentiation phenotype.IAST did not form clone in soft agar and no tumor was developed in nude mice. Conclusion IAST immortalized by SV40Tag proliferates vigorously in vitro and does not belong to malignant transformed cells, so IAST could be used safely in transgenic cellular analgesia.

关 键 词：星形细胞 多瘤病毒转化抗原 猴病毒40 细胞系 细胞周期 慢性疼痛 晚期癌痛 治疗 

分 类 号：R346[医药卫生—基础医学]