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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 cell line (IAST) that controls enkephalin expression by doxycycline (Dox). Methods Recombinant plasmid pRevTREhPPE and regulatory plasmid pRevTet-On were transfected into retroviral packaging cell PT67 by liposome-mediated method. LAST containing RevTet-On and RevTRE / hPPE virus supernatant were infected with LAST to obtain stable expression of enkephalin Real-time quantitative PCR was used to detect the expression of hPPE gene by quantitative real-time PCR. The expression of hPPE gene in this cell line was detected by immunocytochemistry and radioimmunoassay. Peptide expression. Results The expression of hPPE gene and the secretion of enkephalin in IAST / Tet-On / hPPE cell lines were regulated by Dox. When Dox concentration was 100-5000 ng / ml, with the increase of Dox concentration, the expression of hPPE gene and enkephalin Peptide secretion increased with a peak Dox concentration of 5000 ng / ml. Conclusion IAST was successfully constructed by tetracycline and its derivatives doxycycline quantitatively regulating the expression of enkephalin.