RESUMEN
<p><b>OBJECTIVE</b>To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.</p><p><b>METHODS</b>Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay.</p><p><b>RESULTS</b>IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.</p><p><b>CONCLUSION</b>An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.</p>
Asunto(s)
Animales , Ratas , Antibacterianos , Farmacología , Astrocitos , Línea Celular Transformada , Enfermedad Crónica , Doxiciclina , Farmacología , Encefalinas , Genética , Metabolismo , Terapia Genética , Métodos , Vectores Genéticos , Neurotransmisores , Genética , Metabolismo , Manejo del Dolor , Precursores de Proteínas , Genética , Metabolismo , Retroviridae , GenéticaRESUMEN
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 supernatant 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 detected by real time-PCR,immuno- cytochemistry 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 astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.