简介:现在是冬天,早晨非常冷。屋外全是白色的,因为昨天下雪了。狗先生和猫小姐很早起床。早餐后,狗先生想看电视。但是猫小姐说:'噢,亲爱的.外面好漂亮。让我们去堆雪人。'堆雪人可不容易,所以他们非常卖力。哦,多么漂亮的雪人.狗先生把他的帽子截在雪人的头上。在那之后,他们非常饿。他们的午餐是鱼,一些骨头和一个大蛋糕。狗先生说:'都中午了,雪人一定饿了。让我们把蛋糕给他。''好吧.'猫小姐说。
简介:AIM:Totransfectthecatcornealendothelialcells(CECs)withrecombinanthumanβ-nervegrowthfactorgeneadeno-associatedvirus(AAV-β-NGF)andtoobservetheeffectoftheexpressedβ-NGFproteinontheproliferationactivityofcatCECs.METHODS:TheendotheliumofcatcorneawastornunderthemicroscopeandrapidlycultivatedinDulbecco’smodifiedEagle’smedium(DMEM)toformsinglelayerCECsandthepassage2endothelialcellswereusedinthisexperiment.TherecombinanthumanAAV-β-NGFwasconstructed.TherecombinanthumanAAV-β-NGFwastransferredintocatCECsdirectly.Threegroupswereasfollowing:normalCECcontrolgroup,CEC-AAVcontrolgroupandrecombinantCECAAV-β-NGFgroup.Forty-eighthoursaftertransfection,thetotalRNAwasextractedfromtheCECbyTrizol.Theexpressionoftheβ-NGFtargetgenedetectedbyfluorescencequantitativepolymerasechainreaction;proliferationactivityofthetransfectedCECdetectedat48hbyMTTassay;thepercentageofG1cellsamongCECsaftertransfectwasdetectedbyflowcytometrymethod(FCM);cellmorphologywasobservedunderinvertedphasecontrastmicroscope.RESULTS:Thetornendotheliumculturetechniquerapidlycultivatedsinglelayercatcornealendothelialcells.Theself-designedprimersforthetargetgeneandreferencegenewereefficientandspecialconfirmedthroughelectrophoresisanalysisandDNAsequencing.Forty-eighthoursaftertransfect,thehumanβ-NGFgenemRNAdetectedbyfluorescencequantitativepolymerasechainreactionshowedthattherewasnosignificantdifferencebetweennormalCECcontrolgroupandCECAAVcontrolgroup(P>0.05);therewassignificantdifferencebetweentwocontrolgroupsandrecombinantCEC-AAV-β-NGFgroup(P<0.05).MTTassayshowedthattransfectofrecombinantAAV-β-NGFpromotedtheproliferationactivityofcatCEC,whiletherewasnosignificantdifferencebetweennormalCECcontrolgroupandCEC-AAVcontrolgroup(P>0.05).FCMresultshowedthatthepercentageofG1cellsinCECAAV-NGFgroupwas76.8%whi