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21420170208ChineseJournalofTissueEngineeringResearchFebruary8,2017Vol.21,No.4P.O.Box10002,Shenyang110180:B:2095-4344(2017)04-00512-082016-12-29SuMin-hong,Studyingfordoctorate,Physician,DepartmentofRespiratoryMedicine,theFifthAffiliatedHospitalofSunYat-senUniversity,Zhuhai519000,GuangdongProvince,ChinaCorrespondingauthor:HuangJin,DepartmentofRespiratoryMedicine,theFifthAffiliatedHospitalofSunYat-senUniversity,Zhuhai519000,GuangdongProvince,China12311111(151900025100003510630).[J].201721(4):512-519.DOI:10.3969/j.issn.2095-4344.2017.04.004ORCID:0000-0002-3933-2534()C57BL/6702(A)35mg/kg2/81(B)35mg/kg1/882468105Masson8246810A26810B26A(P0.05)Massonβ1αAa-SMAβ1268B24a-SMAβ1A(P0.05)6A(P0.05)1235mg/kgβ1(81470220)A35mg/kg28MassonTGF-β1α-SMACol-18246810AB35mg/kg18.ISSN2095-4344CN21-1581/RCODEN:ZLKHAH513(1DepartmentofRespiratoryMedicine,theFifthAffiliatedHospitalofSunYat-senUniversity,Zhuhai519000,GuangdongProvince,China;2DepartmentofUrology,theSunYat-senMemorialHospital,SunYat-senUniversity,Guangzhou510000,GuangdongProvince,China;3DepartmentofRespiratoryMedicine,theThirdAffiliatedHospitalofSunYat-senUniversity,Guangzhou510630,GuangdongProvince,China)AbstractBACKGROUND:Thereisnoeffectivedrugforidiopathicpulmonaryfibrosis(IPF),becauseofalackoftheanimalmodelimitatingthecompletepathogenesisofhumanIPF.Therefore,itiscriticaltoestablishanidealanimalIPFmodelusedforinvestigatingtheunderlyingpathogenesisanddevelopingakindofeffectivedrug.OBJECTIVE:ToestablishananimalmodelthatcanmimicmorecharactersofhumanIPF.METHODS:SeventymaleC57BL/6micewererandomlydividedintotwogroups,followedbysubjectedtotheintraperitonealinjectionofbleomycin(35mg/kg)ondays1,4,8,11,15,18,22,and25,twice(groupA)oronce(groupB)aweek.Miceweresacrificedat2,4,6,8,and10weeksaftertheeighthinjection,andthelungtissuesweremovedusedforhematoxylin-eosin,Massonandimmunohistochemicalstainings.RESULTSANDCONCLUSION:Therewerevariousdegreesofalveolitisandpulmonaryfibrosisinthetwogroupsatdifferenttimepointsafterthelastinjection.ThescoresofalveolitisandpulmonaryfibrosisinthegroupAbegantograduallyincreasefromthe2ndweekandreachedthehighestlevelatthe6th8thweeksuntilthe10thweek.Incontrast,thescoresofalveolitisandpulmonaryfibrosisinthegroupBpeakedatthe2ndweek,thenfluctuatelydecreased,andweresignificantlylowerthanthoseinthegroupAatthe6thweek(P0.05).ImmunohistochemistryshowedthattypeIcollagendepositionwasmainlydistributedinthesubpleuralregion,peri-vascularregionandalveolarsepta,whichwasconsistentwithMassonstainingfindings.Theexpressionlevelsoftransforminggrowthfactorβ1(TGF-β1)andα-smoothmuscleactin(α-SMA)intheregionsdevelopingalveolitisandpulmonaryfibrosisweresignificantlyincreased.InthegroupA,theexpressionlevelsoftypeIcollagen,TGF-β1,α-SMA,andthehydroxyprolinecontentinthelungtissuesreachedthepeaklevelat68weeks.However,inthegroupB,allaboveindicatorsreachedthehighestlevelatthe2ndweek,butgraduallydecreasedthereafter.Atthe4thweek,theexpressionLevelsofTGF-β1andα-SMAinthegroupBweresignificantlylowerthanthoseinthegroupA(P0.05).Atthe6thweek,thehydroxyprolineandtypeIcollagenlevelsinthegroupBweresignificantlylowerthanthoseinthegroupA(P0.05).Inconclusion,themousemodelofpulmonaryfibrosisinducedbyintraperitonealinjectionof35mg/kgbleomycintwiceweeklycanbeusedtomimictherepetitivewoundhealingprocess,pathologicalmorphologyandcytokinechangesofhumanIPF,whichispronetoadministration,withbetterstabilityandrepeatability.ThismodelisofgreatsignificanceforthestudyonIPF.Subjectheadings:DiseaseModels,Animal;PulmonaryFibrosis;BleomycinFunding:theNationalNaturalScienceFoundationofChina,No.81470220Citethisarticle:SuMH,JiangN,LiHT,WangZG,XieYF,ZhengXB,TuCL,HuangJ.Intraperitonealinjectionofbleomycininducespulmonaryfibrosisinmice:along-termstabilityevaluation.ZhongguoZuzhiGongchengYanjiu.2017;21(4):512-519.0Introduction3566[1-4][35][6-8][9]C57BL/6[10-11]6[12]Chung[12]36Degryse[13]2116[14-16][17]6.P.O.Box10002,Shenyang110180()2011-0029SPFSYXK()2013-01283RSPFSPF222455%60%12h(BLM)15mg/()Masson()I(Col-1)(Abcam)β1(TGF-β1)(Abcam)α-(α-SMA)(Abcam)DAB(Boster)(HYP)(Sigma)(Agilent)1.41.4.1681923g70AB82468105Masson(β1α-SMA)1.4.21.4.38246810610%MassonSzapiel[18]0120%220%50%350%Hubner[19]0132333410%510%50%650%7581.4.4Chen[20]10200()105%05%25%125%50%250%75%375%40()1()2()31.4.580[21]1.5Massonβ1α-SMA1.6SPSS19.0x_±st2P0.052Results2.17022.2A35mg/kg200μL2/8B35mg/kg200μL1/8.ISSN2095-4344CN21-1581/RCODEN:ZLKHAH515(1)A822.86%B1028.57%2.32.4MassonA210Masson()10B2MassonA4810(23)A21068B2A(P=0.580)B4BA(P0.05)10B(4)2.524P0.7320.825AB6AB(P=0.027)(4)2.6Iβ1α-SMA(5)β1(6)α-SMA(7)β1α-SMA2A6810B410(8)3Discussion[11][9]()()[11-22][112225][1122][9]C57BL/6[26][27-30]302520