强力霉素调控表达脑啡肽的永生化大鼠星形胶质细胞株的构建  

Construction of an immortalized rat astrocyte strain with inducible enkephalin expression

作　　者：徐颖[1] 田玉科[1] 田学愎[1] 梅伟[1] 安珂[2] 杨辉[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 [2]中山大学附属第一医院麻醉科

出　　处：《中华麻醉学杂志》2006年第6期534-537,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助课题(30170905);杨森基金;湖北省卫生厅科研基金(Jx2B07)

摘　　要：目的构建受强力霉素(Dox)调控表达脑啡肽的永生化大鼠星形胶质细胞株(IAST)。方法采用脂质体介导法将重组质粒pRevTREhPPE和调节质粒pRevTet-On分别转染逆转录病毒包装细胞PT67；将含有RevTet-On和RevTRE/hPPE病毒上清感染LAST,得到稳定表达脑啡肽的IAST/Tet-On/ hPPE细胞株,实时定量PCR检测Dox定量调控该细胞株前脑啡肽原(hPPE)基因的表达,免疫细胞化学及放射免疫分析法检测Dox定量调控该细胞株中脑啡肽的表达。结果IAST/Tet-On/hPPE细胞株中,hPPE基因的表达和脑啡肽的分泌受Dox调控,Dox浓度为100～5000 ng/ml时,随着Dox浓度增高,hPPE基因的表达和脑啡肽的分泌增加,Dox浓度为5000 ng/ml时达峰值。结论成功构建了受四环素及其衍生物强力霉素定量调控表达脑啡肽的IAST。Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline. Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods. The superuatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain （IAST） and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell, Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On /hPPE cell strain was detecled by real time-PCR, immunocytochemistry and radio-immunoassay. Results Real time-PCR, immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner. Conclusion An immortalized rat astroeyte strain which secrets enkephalin as controlled by doxyeycline has been constructed successfully.

关 键 词：脑啡肽类 多西环素 基因表达调控 星形细胞 

分 类 号：R96[医药卫生—药理学]