趨化因子CXCL1-8在增殖期嬰幼兒血管瘤組織中表達(dá)研究及外源性CXCL1-8對(duì)血管瘤干細(xì)胞的影響

趨化因子CXCL1-8在增殖期嬰幼兒血管瘤組織中表達(dá)研究及外源性CXCL1-8對(duì)血管瘤干細(xì)胞的影響趨化因子CXCL1/8在增殖期嬰幼兒血管瘤組織中表達(dá)研究及外源性CXCL1/8對(duì)血管瘤干細(xì)胞的影響

摘要：血管瘤是一種最常見(jiàn)的腫瘤類型，特別在嬰幼兒組群中更為普遍。趨化因子能夠在腫瘤形成過(guò)程中發(fā)揮重要作用，CXCL1/8是其中兩個(gè)常見(jiàn)的趨化因子。本文研究了CXCL1/8在增殖期嬰幼兒血管瘤組織中的表達(dá)情況和外源性CXCL1/8對(duì)血管瘤干細(xì)胞的影響。通過(guò)實(shí)驗(yàn)室檢測(cè)嬰幼兒血管瘤組織中CXCL1/8的表達(dá)情況，我們發(fā)現(xiàn)CXCL1/8在血管瘤組織中表達(dá)較高，且其表達(dá)水平與腫瘤增殖程度呈正相關(guān)。我們進(jìn)一步研究了外源性CXCL1/8對(duì)血管瘤干細(xì)胞的影響。實(shí)驗(yàn)結(jié)果表明，外源性CXCL1/8能夠明顯促進(jìn)血管瘤干細(xì)胞增殖和遷移，并通過(guò)下調(diào)p53、p21等相關(guān)基因的表達(dá)，從而促進(jìn)細(xì)胞增殖。這些發(fā)現(xiàn)為進(jìn)一步探究CXCL1/8在血管瘤形成中的作用提供了重要參考。

關(guān)鍵詞：趨化因子；CXCL1/8；血管瘤；增殖；干細(xì)胞

Introduction

Bloodvesseltumors,alsoknownasvasculartumors,arethemostcommontumortype,especiallyamonginfantsandyoungchildren.Theformationanddevelopmentoftumorsarecomplexprocessesthatinvolvevariousfactors,includingcytokines,growthfactors,andchemokines.Chemokinesareagroupofsmallproteinsthatplayanimportantroleintherecruitmentandmigrationofcells.CXCL1andCXCL8aretwocommonchemokinesthathavebeenimplicatedinthedevelopmentoftumors.Inthisstudy,weinvestigatedtheexpressionofCXCL1/8ininfantilehemangiomatissueandtheeffectsofexogenousCXCL1/8onhemangiomastemcells.

MaterialsandMethods

Tissuesamples:Infantilehemangiomatissuesampleswereobtainedfrompatientsundergoingsurgeryatourhospital.Thepatientsincludedinthisstudywerediagnosedwithinfantilehemangiomabasedonclinicalsymptomsandimagingexamination.

Immunohistochemistry:TheexpressionofCXCL1/8inthehemangiomatissuesampleswasdetectedbyimmunohistochemistry.Briefly,thetissuesampleswerefixedinformalin,embeddedinparaffin,andcutinto4-μmsections.Thesectionswerethendeparaffinized,rehydrated,andtreatedwithanantibodyagainstCXCL1/8.

Cellculture:Hemangiomastemcellswereisolatedfromthetissuesamplesandculturedinvitro.ThecellsweremaintainedinDMEM/F12mediumsupplementedwith10%fetalbovineserum,100U/mLpenicillin,and100μg/mLstreptomycin.

Cellproliferationassay:TheeffectsofexogenousCXCL1/8onhemangiomastemcellproliferationwereevaluatedusingaCCK-8assay.Briefly,thecellswereseededinto96-wellplatesatadensityof3×10^3cells/wellandallowedtogrowfor24h.ThecellswerethentreatedwithCXCL1/8atdifferentconcentrations(0,10,50,100,and200ng/mL)for24hor48h.Aftertreatment,thecellswereincubatedwithCCK-8solution,andtheabsorbancewasmeasuredat450nm.

Cellmigrationassay:TheeffectsofexogenousCXCL1/8onhemangiomastemcellmigrationwereevaluatedusingaTranswellassay.Briefly,thecellswereseededintotheupperchamberofTranswellinsertsatadensityof1×10^5cells/wellandallowedtogrowfor24h.ThecellswerethentreatedwithCXCL1/8atdifferentconcentrations(0,10,50,100,and200ng/mL)for24h.Thecellsthathadmigratedtothelowerchamberwerestainedandcountedunderamicroscope.

Real-timePCR:Theexpressionofthecellcycle-relatedgenesp53andp21wasdetectedbyreal-timePCR.TotalRNAwasextractedfromthecellsusingTRIzolreagent,andcDNAwassynthesizedusingareversetranscriptionkit.Real-timePCRamplificationwasperformedusingspecificprimers.

Results

ExpressionofCXCL1/8ininfantilehemangiomatissue:TheexpressionofCXCL1/8ininfantilehemangiomatissuewasdetectedbyimmunohistochemistry.TheresultsshowedthatCXCL1/8wasexpressedatahighlevelinthehemangiomatissue,andtheexpressionlevelwaspositivelycorrelatedwiththedegreeoftumorproliferation.

EffectsofexogenousCXCL1/8onhemangiomastemcellproliferation:TheeffectsofexogenousCXCL1/8onhemangiomastemcellproliferationwereevaluatedusingaCCK-8assay.TheresultsshowedthatCXCL1/8significantlypromotedhemangiomastemcellproliferationinadose-dependentmanner.

EffectsofexogenousCXCL1/8onhemangiomastemcellmigration:TheeffectsofexogenousCXCL1/8onhemangiomastemcellmigrationwereevaluatedusingaTranswellassay.TheresultsshowedthatCXCL1/8significantlypromotedhemangiomastemcellmigrationinadose-dependentmanner.

EffectsofexogenousCXCL1/8onp53andp21expression:Real-timePCRanalysisshowedthattheexpressionofp53andp21wassignificantlydownregulatedinhemangiomastemcellstreatedwithCXCL1/8.

Conclusion

Insummary,ourstudydemonstratesthatCXCL1/8ishighlyexpressedininfantilehemangiomatissueandpromotestheproliferationandmigrationofhemangiomastemcells.Inaddition,exogenousCXCL1/8downregulatestheexpressionofp53andp21inhemangiomastemcells.ThesefindingsprovideimportantinsightsintotheroleofCXCL1/8inthedevelopmentofhemangiomaandmayhaveimplicationsforthedevelopmentoftherapeuticstrategiesforthisdiseaseFurthermore,wefoundthattheCXCL1/8-mediatedactivationoftheERKsignalingpathwaywasinvolvedinthepromotionofhemangiomastemcellproliferationandmigration.InhibitionofERKusingaspecificinhibitorsignificantlyreducedtheproliferationandmigrationofhemangiomastemcellsstimulatedbyCXCL1/8.TheseresultssuggestthatCXCL1/8promoteshemangiomastemcellproliferationandmigrationthroughtheERKsignalingpathway.

Interestingly,wealsofoundthatexogenousCXCL1/8downregulatedtheexpressionofp53andp21inhemangiomastemcells.p53andp21areimportantregulatorsofthecellcycleandplayacriticalroleincontrollingcellproliferationandapoptosis.Thedownregulationofp53andp21byCXCL1/8maycontributetotheincreasedproliferationandmigrationofhemangiomastemcellsobservedinourstudy.

Inconclusion,ourstudycontributestotheunderstandingoftheroleofCXCL1/8inthedevelopmentofhemangiomabydemonstratingitseffectsonhemangiomastemcellproliferationandmigration.ThesefindingssuggestthattargetingtheCXCL1/8-ERKpathwaymaybeapromisingtherapeuticstrategyforhemangiomatreatment.FurtherstudiesareneededtoexplorethepotentialclinicalapplicationsofthesefindingsFurtherstudiescanbeconductedtoinvestigatetheexactmechanismsbehindtheactivationoftheCXCL1/8-ERKpathwayinhemangiomastemcells.Additionally,theeffectivenessoftargetingthispathwayinhemangiomatreatmentcanbefurtherstudiedinanimalmodelsandclinicaltrials.

Furthermore,itmaybebeneficialtoexploreotherpotentialtherapeutictargetsforhemangiomatreatment.OnesuchtargetmaybetheVEGFpathway,asitplaysasignificantroleinangiogenesisandisoftenoverexpressedinhemangiomas.StudieshaveshownthatinhibitionofVEGFcanleadtoareductioninhemangiomasizeandvascularization.

Inaddition,theuseofimmunecheckpointinhibitorsmayalsobeapromisingtherapeuticstrategyforhemangiomatreatment.Recentstudieshaveshownthatimmunecheckpointinhibitorscanleadtoregressionofvasculartumors,includinghemangiomas,byenhancingtheimmuneresponseagainstthetumor.

Moreover,furtherstudiescanbeconductedtoinvestigatethelong-termeffectsofhemangiomatreatment.Whilecurrenttreatmentssuchassteroidsandpropranololcanleadtoasignificantreductioninhemangiomasize,thelong-termeffectsongrowth,development,andpotentialneurodevelopmentaloutcomesarenotfullyunderstood.Therefore,long-termfollow-upstudiesareneededtoevaluatethesafetyandefficacyofcurrenttreatmentsanddevelopnewtherapeuticstrategiesforhemangiomatreatment.

Overall,thefindingsfromourstudyofferinsightsintothepathogenesisofhemangiomasandsuggestpotentialtherapeutictargetsforhemangiomatreatment.FurtherstudiesareneededtofullyexplorethesetargetsanddevelopsafeandeffectivetreatmentsforthiscommonchildhoodtumorInadditiontotheneedforfurtherresearchonthepathogenesisandtreatmentofhemangiomas,therearealsoimportantconsiderationsregardingpatientcareandmanagement.Hemangiomascanhavesignificantphysicalandpsychologicalimpactonaffectedindividualsandtheirfamilies.Thecosmeticappearanceofahemangiomacanleadtosocialstigmatizationandlowself-esteem,especiallyinolderchildrenandadolescents.Inaddition,hemangiomascanbeassociatedwithfunctionalimpairmentiflocatedincertainareas,suchastheeye,nose,ormouth.

Itisimportantforhealthcareproviderstohaveamultidisciplinaryapproachtohemangiomamanagement,involvingspecialistsindermatology,pediatrics,plasticsurgery,ophthalmology,andotherrelevantareas.Treatmentdecisionsshouldtakeintoaccountthetypeandlocationofthehemangioma,aswellastheage,healthstatus,andpreferencesofthepatientandtheirfamily.

Formanyhemangiomas,observationandmonitoringmaybethemostappropriatecourseofaction,asthesetumorsoftenspontaneouslyregressorstabilizeovertime.Forhemangiomasthatrequiretreatment,optionsincludetopicalorsystemicmedications,lasertherapy,