人体胰腺癌细胞株PANC.docx
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人体胰腺癌细胞株PANC.docx
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人体胰腺癌细胞株PANC
人体胰腺癌细胞株PANC
【摘要】 [目的]在人体胰腺癌细胞株PANC-1中建立一个四环素可调控周期素D1表达系统,研究抑制周期素D1对胰腺癌细胞的影响。
[方法]反义周期素D1质粒通过两次稳定转染进入胰腺癌细胞株PANC-1细胞中,其表达调控系统采用Tet-Off系统。
通过抑制周期素D1表达对PANC-1细胞生长、集落形成能力以及周期素蛋白表达的影响,评价此系统的可调控性和有效性。
[结果]通过第一次转染pTet-Off质粒,选择两个最佳表达克隆行第二次转染pTRE-反义周期素D1质粒,并通过免疫印记测定挑选出能在Tet-Off系统中最有效地表达反义周期素D1的克隆。
通过Tet-Off系统对反义周期素D1的调控,发现周期素D1的表达抑制可明显地抑制胰腺癌细胞生长和集落能力,并可导致胰腺癌细胞形态学改变。
其抑制作用与四环素调控浓度和时间有关。
[结论]此研究在PANC-1胰腺癌细胞株中建立了一个高效、可诱导的反义周期素D1的表达系统。
通过这个系统的建立,可进一步在体内和体外研究周期素D1的抑制对胰腺癌细胞的影响,并可结合其它治疗手段如化疗来探讨联合治疗在临床的潜在应用价值。
【关键词】周期素D1 反义Tet-Off系统 胰腺肿瘤 PANC-1
Pancreaticcanceristhefourthtofifthleadingcauseofcancerdeathintheworldwithanoverall5-yearsurvivalofonly%[1].Overexpressionofgrowthfactorsinconjunctionwithderegulatedcyclin
D1expressionhasbeenproposedtocontributetothe
lossofcellcyclecontrolandtoenhancetumorigenesis.Astudyof82pancreaticcancersdemonstratedoverexpressionofcyclinD1proteinin65%ofthetumors,andthiswasassociatedwithshortersurvivalforthesepatients.InanotherstudypatientswithhighlevelsofcyclinD1mRNAhadamediansurvivalof months,whereaspatientswithlowlevelshadamediansurvivalof months.
TheaimofthisprojectwastoestablishhumanpancreaticcancercellsthatharborCD1antisensecDNAinatetracyclineinduciblevectorsystemthatcanbeturnedonandoffdependingonthesurroundingtetracyclineconcentration.AndwiththesecellstheeffectsofinhibitionofexpressionofCD1werealsoexplored.
1MATERIALANDMETHODS
Material
PANC-1humanpancreaticcancercellsfromAmericanTypeCultureCollection;pTet-Off,pTRE-LucandpTK-HygfromClontechLaboratoriesInc;plasmidmaxikitandQIAprep@SpinminiprepKitfromQiagen;lipofectaminefromGibco;luminometerandthedual-luciferasereporterassaysystemfromPromega;Mousenonoclonalanti-cyclinD1antibody(DCS-6)fromNeomarkerInc;anti-mouseIgGhorseradishperoxidaselinkedwholeantibody(fromsheep),anti-β-actinantibody;electrochemiluminescence(ECL)solutionandHybond-PmembranesfromAmershamBiosciencesGermany;DNAladder(1Kbplus),pH5αTMchemicallycompetentcellsfromInvitrogen.
Methods
CellCulture
PANC-1cellsweregrowninDMEsupplementedwith10%fetalbovineserum,penicillinG(100units/ml),andstreptomycin(100mg/ml)termedcompletemedium.Cellsweremaintainedinmonolayercultureat37℃inhumidifiedairwith5%CO2.Themediumforcelllinescontainiganeomycinresistancegenewassupplementedwith mg/mlG418and mg/mlforcontainingahygromycinresistancegeneandtermedselectionmedium.DMEsupplementedwith%BSA,5mg/Ltransferrin,5mg/Lselenium,penicillinG(100units/ml),andstreptomycin(100mg/ml)termedserumfreemedium.
TransformationandPlasmidPreparation
ThepTet-Offsystemconsistedofthefollowingplasmids:
aregulatorplasmid(pTet-Off),aluciferaseresponseplasmid(pTRE-Luc),ahygromycinresistantplasmid(pTK-Hyg)andaresponseplasmidincludingfull-lengthhumancyclinD1antisensecDNA(pTRE-CD1AS).
E.colicells(DH5α)wereusedfortransformationwithplasmids.Aftercolonyformationminipreparationswereperformedfirst,followedbymaxipreparationsaftercertificationoftheauthenticityoftheplasmidwithrestrictionendonucleasedigestsasshownforpTet-Off,pTRE-LucandpTK-Hyg,respectively.
TopreparedpTRE-CD1ASthe EcoRI/BamHIfull-lengthhumancyclinD1cDNAwassubclonedinitsantisenseorientationintopTREvector.
Transfection
FirstStableTransfectionwithpTet-OffVector
Cellswereculturedin10cmdishestoadensityof70%~80%.Then40mllipofectamineand40mgpTet-OffplasmidDNAwasaddedtotwotubeswith3mlserumfreemediumseparately.Thetwosolutionswerehomogenizedbeforetheywerethenmixedtogether.Themixturewasthenincubatedfor30minutesatroomtemperature.Cellswerewashedinthemeantimetwotimeswith1×PBSandonetimewithserumfreemedium.Then,thehomogenizedmixtureoflipofectamineandthepTet-OffplasmidDNAwereaddedtothecellswhichwereincubatedfor5hoursat37℃.Afterthatanequalvolumeofcompletemediumcontaining20%FBSwasaddedforanadditional20hours.Next,cellswerewashedtwotimeswith1×PBSandtwotimeswithcompletemedium.Afterincubatingforanother24hours,cellsweresplitintoeight10cmdishesandincubatedinselectionmedium(/mlG418).Selectionmediumwaschangedweekly.After2weekscoloniesformed.Thevisiblecolonieswerepickedindividuallywithsterilecottontipswettedwithtrypsin.Thepickedcoloniesweretransferredinto24-wellplatesincluding1mlselectionmediumperwell.
TransientTransfectionwithpTRE-Luc
Cellswereculturedin10cmdishestoadensityof70%~80%.Onthefollowingday,40mllipofectamineand40mgpTRE-LucplasmidDNAwasaddedtoseparatetubeswith3mlserumfreemedium.Thetwosolutionswerehomogenizedbeforetheyweremixedtogethertomakethetransfectionsolution.Themixturewasthenagainincubatedfor30minutesatroomtemperature.Cellswerewashedtwotimeswith1×PBSandonetimewithserumfreemedium.Themixturewashomogenizedagainandaddedtothecells.Cellswerethenincubatedovernightat37℃.Onthefollowingday,cellswerewashedonetimewith1×PBSandtransferredinto24-wellplateswithcompletemedium(everycloneinto4wells).Tetracycline(2mg/ml)wasaddedto2ofthe4wellsofeachclone.Cellswerethenincubated24hoursat37℃andcollectedforluciferaseactivityassay.
SecondStableCo-transfectionwithpTRE-cyclinD1AntisenseandpTRE-Hyg
Cellswereculturedin10cmdishestoadensityof70%~80%.Afterthat40mllipofectamine,40mgpTRE-CD1ASand2mgpTRE-HygplasmidDNAweremixedandincubatedin6mlserumfreemediumfor30minutesatroomtemperature.Cellswerethenwashedtwotimeswith1×PBS,onetimewithserumfreemediumandincubatedwiththetransfectionsolutionovernightinthepresenceoftetracycline(2μg/ml).Onthenextday,cellswerewashedtwotimeswith1×PBSandtwotimeswithcompletemediumandthenincubatedwithcompletemediuminthepresenceoftetracycline(2μg/ml).
Cellswereallowedtogrowtoabout80%confluenceandthensplitintoeight10cmdishesfollowedbyincubationwithcompletemediumincludinghygromycin(/ml)andtetracycline(2μg/ml).Mediumwaschangedonceaweekincludingtherespectiveaddition.After4weekscolonieshadformedandwerepickedindividuallyasdescribedabove.Coloniesweretransferredinto24-wellplatesin1mlselectionmediumincludingtetracycline(2μg/ml).WhencellsreachedconfluencetheyweretransferredintoflasksinselectionmediumincludingG418(/ml),hygromycin(/ml)andtetracycline(2μg/ml).
Dual-luciferaseReporterAssay
CellswerecollectedandprocessedaccordingtheprotocoldescribedwithDual-LuciferaseReporterAssaySystemkit.Aluminometerwasusedtomeasuretheactivity.
MTT-Assay
75,000cells/mlwereseededin200mlcompletemediumin96-wellplates.Onthenextday,mediumwasremovedwithoutremovingcells.Cellswerewashedwith1×PBS(200ml)andthen200mlmediuminabsenceorpresenceofdesiredadditiveswasaddedtoeachwell.Every24hoursmediumwaschanged.Toinitiatetheassay,25mlMTTstock-solution(5%)wasaddedineachwell.Cellswereincubatedforadditional4hourspriortotheremovalofthemedium.Thedevelopeddyecrystalsweredissolvedin100mlacidicisopropanol(isopropanolwith Nhydrogenchloride)for15minutes.Afterthat,theopticaldensityvaluesweremeasuredusingamicroplatereaderat570nm.Thevalueat650nmwassubtractedasbackground.
SoftAgar-assay
MediumA(15%fetalbovineserum,1mMsodium-pyruvate,8μg/mlL-serine,100units/mlpenicillin,100μg/mlstreptomycin,11%trypticsoybroth,16μg/mlL-asparaginewithMcCoys′5A,)andmediumB(15%fetalbovineserum,2μg/mlinsulin,1%vitamineC,100units/mlpenicillin,100μg/mlstreptomycin,2mML-glutamine,1%L-asparaginewithRPMImedium)werepreparedfirst.1%agarsolutionwasmeltedbymicrowavingandaddedtomediumA(1∶5v/v)tomakebaseagar.Then,1mlwaspouredintoperwellof dishesandkeptatroomtemperatureuntilitbecamesolid.
Inthesametime,cellsweretrypsinizedanddilutedto×105cells/mlinmediumB.Cellswerethenincubatedinawaterbathat37°Cand1%agarsolution(50°C)wasaddedtothecellsuspension(1∶v/v)includingthedesiredtreatmentagents,mixedandpoured(1mlperwell)asupperagar.Latertheagarplateswereincubatedat37℃inhumidifiedairwith5%CO2.After5days,cellswerefedwith feedingsolution(1%agar:
mediumB1∶2(v/v))perwell.After10~14days50μl5%MTTsolutionwasaddedtoeachplate.Thenextdaystainedcolonieswerecountedunderamicroscopeandphotographed.
Immunoblotting
Exponentiallygrowingcells(40%~50%confluent)werelysedandproteinconcentrationofeachcelllysatewaspdeterminedbyBCAProteinAssayReagentkit.Celllysateswerethensubjectedto12%SDS-PAGE,runwith70Vfor30minutesandthenwith160Vfor90minutes.ElectrophoresedproteinswereelectrotransferredfromthegeltoHybond-Pmembranesusing110Vfor60minutes.Afterblockingin5%milkin1×TTBS,themembraneswereblottedwithahighlyspecificmousemonoclonalanti-cyclinD1antibody(DCS-6,1∶500)andwithasecondaryanti-mousehorseradish-conjugatedantibody(1∶1000).Boundantibodieswerevisualizedusingenhancedchemiluminescence.
Toconfirmequalloading,membraneswerestrippedfor30minutesat50℃inbufferandre-probedwithananti-β-actinantibody.
FlowCytometry
Cellswereharvestedbyscrapingandwashedthreetimeswithbuffersolution(cycletestTMplusDNAreagentkit).Cellswerethenlysedandstainedwithpropidiumiodidestainingsolutionaccordingtoinstr
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