新型氯膦酸二钠脂质体灌肠剂诱导小鼠巨噬细胞凋亡
ANovelClodronateLiposomalEnemaInducesApoptosisinMurineMacrophages全文下载:PDF(690KB)HTMLXMLPP.90-98DOI:10.12677/WJCR.2017.74014下载量:746浏览量:2,098科研立项经费支持
目的：探讨一种氯膦酸二钠脂质体灌肠剂的制备及其诱导巨噬细胞凋亡的作用。方法：以DPPC和胆固醇为主要组分并添加带负电荷的磷酸二鲸蜡酯，用薄膜水化法制备脂质体；用MTT法测定该氯膦酸二钠脂质体对离体培养的小鼠巨噬细胞的致死活性；以免疫印迹法和流式细胞术对该氯膦酸二钠脂质体诱导的细胞凋亡进行了研究。结果：薄膜水化法制备所得的脂质体粒径适于巨噬细胞吞噬。用离体培养的小鼠巨噬细胞对氯膦酸二钠脂质体灌肠剂的细胞致死活性进行评估，该氯膦酸二钠脂质体制剂可有效地杀伤巨噬细胞，其细胞毒性随浓度而增加；免疫印迹法可从氯膦酸二钠脂质体处理的细胞提取物中检测到显著增加的活性半胱天冬酶-3，表明氯膦酸二钠脂质体可诱导细胞凋亡；流式细胞术检测AnnexinV的结果确证氯膦酸二钠脂质体可诱导巨噬细胞凋亡。结论：该氯膦酸二钠脂质体制剂可诱导巨噬细胞凋亡从而清除巨噬细胞，有望用于某些免疫性疾病和炎症性疾病的辅助治疗。

Aim:Toinvestigateapoptoticeffectofanovelclodronateliposomalenema.Methods:Anovelclodronateliposomalenemawasdevelopedbythin-filmhydrationusingDPPC,cholesterol,andnegativelychargeddicetylphosphate.LethalityofclodronateliposomeswasdeterminedbyMTTassayusingRAW264.7murinemacrophages.Clodronateliposome-inducedapoptosiswasinvestigatedbyWesternblottingandfluorescence-activatedcellsorting(FACS).Results:Anovelclodronateliposomalenemawasdevelopedbythin-filmhydration.Thisliposomalenemahadasizerangethatissuitableforphagocytosis.MTTassayshowedthatthisclodronateliposomalenemaefficientlycausedcytotoxicityofRAW264.7murinemacrophagesinadose-dependentmanner.Westernblotsshowedremarkablyincreasedcleavedcaspase-3inclodronateliposome-treatedRAW264.7cellscomparedtocontrols,indicatingapoptosisinducedbyclodronateliposomes.Inaddition,FACSanalysisforAnnexinVconfirmedthatclodronateliposomesinducedapoptosisinRAW264.7macrophages.Conclusion:Thisclodronateliposomalenemaiscapableofinducingmacrophageapoptosis,andmaybeusedformacrophagedepletionincertaindiseasesasanadjuvanttherapy.
杨永红,李海波,王兴民.新型氯膦酸二钠脂质体灌肠剂诱导小鼠巨噬细胞凋亡[J].世界肿瘤研究,2017,7(4):90-98.https://doi.org/10.12677/WJCR.2017.74014
vanRooijen,N.,Sanders,A.andvandenBerg,T.K.(1996)ApoptosisofMacrophagesInducedbyLiposome-MediatedIntracellularDeliveryofClodronateandPropamidine.JournalofImmunologicalMethods,193,93-99.
https://doi.org/10.1016/0022-1759(96)00056-7
Merlini,G.andTuresson,I.(1996)UtilityofBisphosphonatesinTreatingBoneMetastases.MedicalOncology,13,215-221.
https://doi.org/10.1007/BF02990934
Dominguez,L.J.,DiBella,G.,Belvedere,M.andBarbagallo,M.(2011)PhysiologyoftheAgingBoneandMechanismsofActionofBisphosphonates.Biogerontology,12,397-408.
https://doi.org/10.1007/s10522-011-9344-5
MufamADI,M.S.,Pillay,V.,Choonara,Y.E.,DuToit,L.C.,Modi,G.,Naidoo,D.andNdesendo,V.M.(2011)AReviewonCompositeLiposomalTechnologiesforSpecializedDrugDelivery.JournalofDrugDelivery,2011,939851.
https://doi.org/10.1155/2011/939851
Li,Y.,Lee,P.Y.andReeves,W.H.(2010)MonocyteandMacrophageAbnormalitiesinSystemicLupusErythematosus.ArchivumImmunologiaeetTherapiaeExperimentalis,58,355-364.
https://doi.org/10.1007/s00005-010-0093-y
Chalmers,S.A.,Chitu,V.,Herlitz,L.C.,Sahu,R.,Stanley,E.R.andPutterman,C.(2015)MacrophageDepletionAmelioratesNephritisInducedbyPathogenicAntibodies.JournalofAutoimmunity,57,42-52.
https://doi.org/10.1016/j.jaut.2014.11.007
Thomas,D.andApovian,C.(2017)MacrophageFunctionsinLeanandObeseAdiposeTissue.Metabolism,72,120-143.
https://doi.org/10.1016/j.metabol.2017.04.005
Bobryshev,Y.V.,Nikiforov,N.G.,Elizova,N.V.andOrekhov,A.N.(2017)MacrophagesandTheirContributiontotheDevelopmentofAtherosclerosis.ResultsandProblemsinCellDifferentiation,62,273-298.
https://doi.org/10.1007/978-3-319-54090-0_11
Qian,B.Z.andPollard,J.W.(2010)MacrophageDiversityEnhancesTumorProgressionandMetastasis.Cell,141,39-51.
https://doi.org/10.1016/j.cell.2010.03.014
Yang,Y.,Wang,X.,Huycke,T.,Moore,D.R.,Lightfoot,S.A.andHuycke,M.M.(2013)ColonMacrophagesPolarizedbyCommensalBacteriaCauseColitisandCancerthroughtheBystanderEffect.TranslationalOncology,6,596-606.
https://doi.org/10.1593/tlo.13412
Popovska,O.(2014)AnOverview:MethodsforPreparationandCharacterizationofLiposomesasDrugDeliverySystems.InternationalJournalofPharmaceuticalandPhytopharmacologicalResearch,3.
Calvagno,M.G.,Celia,C.,Paolino,D.,Cosco,D.,Iannone,M.,Castelli,F.,Doldo,P.andFrest,M.(2007)EffectsofLipidCompositionandPreparationConditionsonPhysical-ChemicalProperties,TechnologicalParametersandInVitroBIOLOGicalActivityofGemcitABIne-LoadedLiposomes.CurrentDrugDelivery,4,89-101.
https://doi.org/10.2174/156720107779314749
Vorauer-Uhl,K.,Wagner,A.,Borth,N.andKatinger,H.(2000)DeterminationofLiposomeSizeDistributionbyFlowCytometry.Cytometry,39,166-171.

Otake,K.,Shimomura,T.,Goto,T.,Imura,T.,Furuya,T.,Yoda,S.,Takebayashi,Y.,Sakai,H.andAbe,M.(2006)PreparationofLiposomesUsinganImprovedSupercriticalReversePhaseEvaporationMethod.Langmuir,22,2543-2550.
https://doi.org/10.1021/la051654u
Krasnici,S.,Werner,A.,Eichhorn,M.E.,Schmitt-Sody,M.,Pahernik,S.A.,Sauer,B.,Schulze,B.,Teifel,M.,Michaelis,U.,Naujoks,K.andDellian,M.(2003)EffectoftheSurfaceChargeofLiposomesonTheirUptakebyAngiogenicTumorVessels.InternationalJournalofCancer,105,561-567.
https://doi.org/10.1002/ijc.11108
Frohlich,E.(2012)TheRoleofSurfaceChargeinCellularUptakeandCytotoxicityofMedicalNanoparticles.InternationalJournalofNanomedicine,7,5577-5591.
https://doi.org/10.2147/IJN.S36111
Gabizon,A.andPapahadjopoulos,D.(1988)LiposomeFormulationswithProlongedCirculationTimeinBloodandEnhancedUptakebyTumors.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,85,6949-6953.
https://doi.org/10.1073/pnas.85.18.6949
Hirota,K.andTerada,H.(2012)EndocytosisofParticleFormulationsbyMacrophagesandItsApplicationtoClinicalTreatment.INTECHOpenAccessPublisher.
https://doi.org/10.5772/45820
Pratten,M.K.andLloyd,J.B.(1986)PinocytosisandPhagocytosis:TheEffectofSizeofaParticulateSubstrateonItsModeofCapturebyRatPeritonealMacrophagesCulturedInVitro.BiochimicaetBiophysicaActa,881,307-313.
https://doi.org/10.1016/0304-4165(86)90020-6
Lee,J.S.,Hwang,S.Y.andLee,E.K.(2015)Imaging-BasedAnalysisofLiposomeInternalizationtoMacrophageCells:EffectsofLiposomeSizeandSurfaceModificationwithPEGMoiety.ColloidsandSurfacesB:Biointerfaces,136,786-790.
https://doi.org/10.1016/j.colsurfb.2015.10.029
Selander,K.S.,Monkkonen,J.,Karhukorpi,E.K.,Harkonen,P.,Hannuniemi,R.andVaananen,H.K.(1996)CharacteristicsofClodronate-InducedApoptosisinOsteoclastsandMacrophages.MolecularPharmacology,50,1127-1138.