简介：Objective:Toprovideasoundcellsourceforfurtherex-vivogenetherapyforchronicpain,weattempttodevelopanimmortalizedratastrocytecelllinethatexpressesenkephalinregulatedbydoxycycline.Methods:Retrovirusinfectionmethodwasemployedtodevelopanimmortalizedratastrocytecelllinethatcouldexpressenkephalinregulatedbydoxycycline.ThehPPEgeneexpressionlevelofimmoralizedastroytecells(IAC)/hPPEwasdetectedbyRT-PCR,indirectimmunofluorescencestainingandradioimmunoassay.Results:IACcarryingTet-onsystemtransfectedwithpreproenkephalingenecouldsecreteenkephalinthatwasregulatedbydoxycyclineinadose-dependentmannerandhPPEgeneactivationcouldberepeatedinon-off-oncyclesthroughadministrationorremovalofdoxycycline.Conclusion:Animmortalizedratastrocytecelllinethatsecreteenkephalinunderthecontrolofdoxycyclineisestablishedsuccessfully,whichprovidesaresearchbasisfortransgeniccelltransplantationforanalgesia.

简介：Objective:ToprovideahighlyefficientadenoviralvectorAd-CMV-hTGFβ1forthestudyofgenetherapyforreversionoftheintervertebraldiscdegeneration.Methods:Anewlydevelopedrecombinantadenoviralvectorconstructionsystemwasusedinthestudy.ThecDNAofhTGFβ1wasfirstsubclonedintoashuttleplasmidpShuttle-CMV.TheresultantplasmidwaslinearizedbydigestingwithrestrictionendonucleasePmeI,andsubsequentlytransformedintoE.coll.BJ5183cellswithanadenoviralbackboneplasmidpAdEasy-1.Recombinantswereselectedbykanamycinresistanceandconfirmedbyrestrictionendonucleaseanalysis.Finally,therecombinantplasmidlinearizedbyPmeIwastransfectedinto293cells.Recombinantadenovirusesweregeneratedwithin2weeks.Results:TherecombinantadenoviralplasmidswerecutbyBamHIandPacIrespectively,andthediagnosticfragmentsappearedin0.8%agaroseelectrophoresis.Theinfected293cellsshowedevidentcytopathlceffect(CPE).TheproductionsofPCRconfirmedthepresenceofrecombinantadenovirus.TheexpressionofhTGFβ1wasverifiedbyimmunohistochemicalstaining.Conclusions:ThesuccessfulgenerationoftheadenoviralvectorAd-CMV-hTGFβ1andtheconfirmationoftheinterestgeneexpressionmakeitpossiblefortheexperimentalstudyofthereversionoftheintervertebraldiscdegenerationbygenetherapy.

简介：摘要：2020年，我国注册护士总数