comConstruction of an immortalized rat astrocyte strain with inducible enkephalin expression

    XU Ying, TIAN Yuª²Ke, TIAN Xueª²Bi, MEI Wei,AN Ke, YANG Hui

    Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,Wuhan 430030,China

    ¡¾Abstract¡¿ 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. pRevTetª²On and pRevTRE/hPPE were separately transfected into packaging cell PT67. The RevTetª²On virus supernatants were used to infect IAST and positive colonies were transiently transfected with pRevTREª²Luc plasmid. One IAST/Tetª²On clone with high inducibility and low background was selected by detecting luciferase activity. RevTRE/hPPE virus was used to infect the IAST/Tetª²On strain. IAST that stably expressed Tetª²regulated enkephalin was established. hPPE gene expression levels of IAST/Tetª²On/hPPE cell strains were detected by RTª²PCR, immunocytochemistry and indirect immunofluorescence staining. RESULTS: A recombinant retroviral vector pRevTRE/hPPE was constructed successfully. The inducibility multiple of the IAST/Tetª²On cell strain was 20.6. RTª²PCR and immunocytochemistry confirmed that enkephalin expression level was regulated by Doxycycline in a doseª²dependent 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. ......