sionelectronmicroscopy.ExosomeMarkerproteinsweredetectedbyWesternblotanalysis.Twopotentialhepatoma-associatedproteins,tissuetransglutaminase2(TGM2)andannexinA2,wereanalyzed.

RESULTS:Theexosomesseparatedbythenewex-tractionassaybasedonthenanomaterialweredisc-shaped,intactvesicleswithlipidbilayermembranes.Theywereapproximately30-100nmindiameter,whichissimilartothediameterofexosomesisolatedbythetraditionalmethod.Theproteinconcentrationofexo-somesextractedbythenewmethodwasapproximately

780μg/108

cells,andtherefore,itwas19timeshigherthanthatofexosomesextractedinthetraditionalman-ner.ThereweredifferencesbetweenthetotalproteinsofHuh-7cellsandtheexosomalproteins.Typicalexo-someproteins,suchasthetransmembraneproteinCD63andheatshockprotein70,wereconfirmed.Twopotentialhepatoma-associatedproteinswerealsoiden-tified.TGM2wasfirstfoundtoexistintheexosomesofhumanlivercancercells,butannexinA2wasnotsecretedintoexosomes.

CONCLUSION:Thenewextractionmethodbasedonthenanomaterialisquickandefficient.Thecancer-associatedproteinTGM2canbesecretedthroughanexosome-mediatednon-classicalsecretionpathway,anditmaybeavaluabletumormarker.

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Keywords:Exosome;Membranevesicles;Tissuetrans-glutaminase2;AnnexinA2;Nanomaterials

Coretip:Thetraditionalextractionassayofexosomesisusuallycomplicatedandtimeconsuming.Inthismanuscript,weinvestigateanovelassaybasedonananomaterialtoextractexosomesmorequicklyandefficientlycomparedwiththetraditionalmethod.Ahepatoma-associatedprotein,tissuetransglutaminase2,wasfirstfoundtoexistintheexosomeofthehuman

Novelmethodforextractingexosomesofhepatocellularcarcinomacells

LinZhu,Xiu-HuaQu,Yu-LinSun,Yang-MingQian,Xiao-HangZhao

LinZhu,ThirdSchoolofClinicalMedicine,SouthernMedicalUniversity,Guangzhou510515,GuangdongProvince,China

Xiu-HuaQu,Yang-MingQian,Xiao-HangZhao,CenterofBasicMedicalSciences,NavyGeneralHospitalofChinesePLA,Beijing100048,China

Yu-LinSun,Xiao-HangZhao,StateKeyLaboratoryofMolecu-larOncology,CancerInstituteandHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing100021,China

Authorcontributions:ZhuLperformedthemajorityoftheworkandwrotethepaper;QuXHandSunYLassistedintheexperimentsanddataanalysis;QianYMandZhaoXHweresu-pervisorsforthiswork;andZhaoXHdesignedthestudy.

SupportedbyGrantsfromtheStateKeyProjectsforBasicRe-search,No.2011CB910703andNo.2012ZX10002-017(toZhaoXH);NationalHigh-techRandDProgram,No.2013AA041201(toQianYM)andNo.2012AA020206(toZhaoXH);NationalNaturalScienceFoundationofChina,No.81372591andNo.81321091(toZhaoXH);andtheResearchFoundationoftheCenterforMarineMedicineandRescueofTsinghuaUniversityandNGH(toQianYMandZhaoXH)

Correspondenceto:Xiao-HangZhao,Professor,CenterofBasicMedicalSciences,NavyGeneralHospitalofChinesePLA,Beijing100048,China.zhaoxh@http://www.wendangku.net/doc/9fbd525d0508763231121298.html

Telephone:+86-10-66951482Fax:+86-10-87778360Received:October31,2013Revised:February8,2014Accepted:March4,2014

Publishedonline:June7,2014

Abstract

AIM:Todevelopanovelmethodfortherapidandef-ficientextractionofexosomessecretedbytumorcells.METHODS:Unlikethetraditionalextractionmethod,thesupernatantsofcellcultureswereconcentrated,andtheexosomeswereisolatedpromptlyandef-fectivelyusinganovelnanomaterialcalledExoQuick.Coomassiebrilliantbluestainingwasusedforproteinquantification,andthemorphologyoftheexosomesextractedbybothmethodswasvisualizedbytransmis-

OBSERVATIONALSTUDY

SubmitaManuscript:http://www.wendangku.net/doc/9fbd525d0508763231121298.html/esps/HelpDesk:http://www.wendangku.net/doc/9fbd525d0508763231121298.html/esps/helpdesk.aspxDOI:10.3748/wjg.v20.i21.6651

WorldJGastroenterol2014June7;20(21):6651-6657ISSN1007-9327(print)ISSN2219-2840(online)

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