可诱导表达脑啡肽的大鼠星形胶质细胞株的构建

目的:构建受强力霉素诱导表达脑啡肽的大鼠星形胶质细胞株(IAST).方法:应用分子克隆技术构建含人前脑啡肽原基因(hPPE)的逆转录病毒四环素反应质粒(pRevTRE/hPPE);以脂质体转染法将重组质粒pRevTRE/hPPE和调节质粒pRevTet-On分别转染入包装细胞PT67,分别收集病毒上清;将含有RevTet-On病毒上清感染IAST,挑选单克隆并瞬时转染报告质粒pRevTRE-Luc,通过检测萤光素酶活性,挑选出强力霉素诱导表达高、背景表达低IAST/Tet-On细胞株;再将RevTRE/hPPE病毒上清感染IAST/Tet-On细胞株,得到稳定表达脑啡肽的IAST/Tet-On/hPPE细胞株,通过RT-PCR、免疫细胞化学及间接免疫荧光染色检测该细胞株中强力霉素定量调控脑啡肽的表达.结果:重组逆转录病毒载体pRevTRE/hPPE构建成功;经挑选得到了高表达、低背景IAST/Tet-On细胞株,其诱导倍数为20.6;在IAST/Tet-On/hPPE细胞株中,hPPE基因表达受强力霉素调控,呈剂量依赖关系.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永生化IAST,为可调控基因治疗慢性疼痛的研究奠定了基础.

Construction of an immortalized rat astrocyte strain with inducible enkephalin expression

AIM: To construct an immortalized rat astrocyte strain (IAST) expressing enkephalin regulated by Doxycycline. METHODS: Human preproenkephalin gene (hPPE) was inserted into the plasmid pRevTRE to construct recombinant retroviral vector pRevTRE/hPPE. pRevTet-On and pRevTRE/hPPE were separately transfected into packaging cell PT67. The RevTet-On virus supernatants were used to infect IAST and positive colonies were transiently transfected with pRevTRE-Luc plasmid. One IAST/Tet-On clone with high inducibility and low background was selected by detecting luciferase activity. RevTRE/hPPE virus was used to infect the IAST/Tet-On strain. IAST that stably expressed Tet-regulated enkephalin was established. hPPE gene expression levels of IAST/Tet-On/hPPE cell strains were detected by RT-PCR, immunocytochemistry and indirect immunofluorescence staining. RESULTS: A recombinant retroviral vector pRevTRE/hPPE was constructed successfully. The inducibility multiple of the IAST/Tet-On cell strain was 20.6. RT-PCR and immunocytochemistry confirmed that enkephalin expression level was regulated by Doxycycline in a dose-dependent manner. CONCLUSION: An IAST which secretes enkephalin under the control of Doxycycline has been established successfully, which provides a good basis for further research on regulated gene therapy for chronic pain.