中文摘要：

目的构建四环素及其衍生物强力霉素诱导表达目的基因的永生化大鼠星形胶质细胞株。方法用脂质体法将逆转录病毒载体四环素调控系统中的质粒pBevTet-On转染病毒包装细胞PT67，经筛选培养后获得病毒载体BevTet-On，采用BT-PCR进行鉴定，并应用NIH3T3细胞测定病毒滴度。将BevTet-On感染永生化大鼠星形胶质细胞，用有限稀释法挑选阳性细胞单克隆后扩大培养，每个克隆瞬时转染含萤光素酶报告基因的质粒pBevTRE-Luc，加入强力霉素48h后分别检测其萤光素酶活性值，挑选出强力霉素诱导表达高、背景表达低的细胞株并检测其诱导表达的时效、量效关系。结果BT-PCR结果显示逆转录病毒包装成功，病毒最高滴度为7．4×10^5CFU／ml。RevTet-On转染永生化大鼠星形胶质细胞后挑选出48个单克隆，瞬时转染pBevTER-Lue后筛选出1株高表达低背景细胞株，其诱导表达值为876．1RLU，背景表达值为42．5RLU，诱导倍数为20．6。该细胞株在加入诱导因子强力霉素1h后目的基因即开始表达，在24h时达到高峰，在强力霉素浓度100—2000ng/ml的范围内， 其诱导表达活性与药物浓度呈剂量依赖性。结论含四环素调控系统的永生化大鼠星形胶质细胞株诱导活性可靠，可用于调控表达外源基因的研究。

英文摘要：

Objective To establish and identify tetracycline controlled gene inducible system in immortalized rat astrocyte strains.Methods The PT67 cells were transfected with pRevTet-On vector. The transfected ceils were selected in medium containing C_,418. RT-PCR assay was used to confirm that Rev Tet-On virus was packed correctly. The viral fiter was assayed by infecting NIH3T3 cells. The immortalized astrocyte was infected by RevTet-On virus and individual clones were selected. All individual clones were transiently transfected with prey TRE-Luc, which carried luciferase gene. The clone with low background and high induction expression was selected and its inducibility was tested.Results The RT-PCR assay showed that RevTet-On virus was packed successfully. The highest viral titer of RevTet-On was 7.4× 10^5 CFU/ml. Rev Tet-On infected immortalized astrecyte and 48 individual clones were isolated, Clone 6 was selected for its highest induction of the luciferase activity in response to doxycycline and the lowest leakiness （activity in the absence of doxycycline） and its inducible fold was 20.6. The expression of luciferase was induced in a dose-dependent manner by doxycycline at the concentrations between 100 and 2 000 ng·ml^-1 . The expression of luciferase began lh after doxycycline administration （1 000 ng·ml^-1） and reached the maximum level 48 h later. Conclusion The tetracycline controlled inducible system is established successfully in immortalized rat astrocyte strains, which is useful for the study of control exogenous gene expression.