心血管相关基因芯片的制备及其在知母皂苷作用机理研究...

1,2,1,2,2,1,13(1.,100850;2.,518026):8780mgL-18hRNA,Cy5Cy3,,,(AGT)2A(ADRA2R)21(ECE21),:;;;:R282.71;Q344.13:A:0513-4870(2003)07-0496-05InvestigationsonthemolecularmechanismsofsaponinsfromAnemarrhenaasphodeloidesBungeusingoligonucleotidemicroarraysLIZe2song1,2,LIDe2liang1,HUANGJian2,DINGYu2,MABai2ping1,WANGSheng2qi13(1.InstituteofRadiationMedicineScience,AcademyofMilitaryMedicalSciences,Beijing100850,China;2.ShenzhenYiShengTangBiologicalProductsCo.Ltd,Shenzhen518026,China)Abstract:AimToinvestigatethemolecularmechanismsofsaponinsfromtherhizomeofAnemarrhenaasphodeloidesBunge.MethodsOligonucleotidemicroarraysconsistingof87probesrepresenting87humancardiovasculardisease2relatedgeneswereconstructed.EffectsofsaponinsongeneexpressioninhumanumbilicalveinendothelialcellswereanalyzedbycomparinghybridizationofCy52labeledcDNAsfromsaponins2treatedhumanumbilicalveinendothelialcellsandCy32labeledcDNAsfromuntreatedhumanumbilicalveinendothelialcells.ResultsTheresultsindicatethatangiotensinogengene,2A2adrenoceptorgeneandendothelin2convertingenzyme1geneweredownregulated218,119and311foldsrespectivelyafterhumanumbilicalveinendothelialcellswereincubatedinmediumcontaining80mgL-1saponins.ConclusionTheseresultssuggestthatsaponinsmayhavebeneficialeffectoncardiovasculardiseasesbymodulatingthefunctionofveinendothialcellsandmicroarraycanbeusedtoinvestigatethebiologicalactionofextractsfromtraditionalChinesemedicine.Keywords:Anemarrhenaasphodeloides;saponins;microarrays;geneexpression:2002208228.:(39889001);(01Z019).3Tel:[phone],Fax:[phone],E2mail:[e-mail],,[1],[2],[3],,,,,,,[4,5]694ActaPharmaceuticaSinica2003,38(7):496-500©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,[6,7]GenBankmRNA,MProbe[8],40nt,GC45%55%,,3BLAST,50%12[9,10][12]ABI8909DNA,53N2MMTr2622222N,N2()[10]55P15h,OPC3SSC015gL-1Pixsys5500,015mm,,012%SDS2,2,()100g,80%400mL,4,,(250mL)2,6,,13g(Ecv2304)10%DMEM,37,5%CO280%,80mgL-1,,8h,RNATrizolRNA,:(PBS)(pH712)2,Trizol1mL,1,RNA,75%2,,DEPC,cDNARNASchena[10,13]:RNA60g,mRNA011g,OligodT(17)1g,,7010min,3L,DTT115L,dNTP115L,Cy32dUTPCy52dUTP115L,,42,2min,115L,422h65NaOHRNA,cDNA10LcDNA,963min,,,,4235h,2SSC+012%SDS,011%SSC+012%SDS,011%SSC5min,ScanArray4000,ImaGene412Cy3Cy51mRNA[11]011g,10,1102,1103,1104,1105,RNA,,(1)1104,;1105,110-4g,1108Figure1Detectionsensitivityofmicroarray.DifferentamountsofluciferasemRNAwasspikedintoHUVECtotalRNA,thencDNAswerelabeledwithCy3byreversetranscription.ThelabeledcDNAswerehybridizedtomicroarraycontainingprobeofluciferase(positivecontrol)25,(0101,011,012,015,110,210,310gL-1)(2,3)794:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.(0101015gL-1),;,,015gL-1Figure2Fluorescenceimageofanarraycontainingdifferentconcentrationsofprobes(0101,011,012,015,110,210,310gL-1)(POTCH,ACTC,MYBPC,CaATP2,iNOS)onthefluorescentsignalintensitiesFigure3Effectsofdifferentconcentrationsofprobes3,5,3(2,rpoB)2()RNACy3cDNAmRNACy3cDNAcDNA4,,cDNAcDNA(4A,B)Figure4Detectionspecificityofmicroarray.ThelabeledcDNAsfromHUVECtotalRNA(A),luciferasemRNA(B),andHUVECtotalRNAandluciferasemRNA(C)werehybridizedtoanarrayincludingricegene,rpoBgene,luciferasegeneandhumangene(KALX,LTA,ALR,IGF21,C2myc)4RNACy3Cy5,33Cy3PCy5Cy5PCy31125,7%;Cy3PCy5Cy5PCy31141,115%;3Cy3PCy5Cy5PCy3116(5)Figure5Comparisonofthreedifferentexperiments5RNACy32dUTP,RNACy52dUTP6Cy32dUTP894ActaPharmaceuticaSinica2003,38(7):496-500©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Cy52dUTPcDNA,,Figure6Pseudocolorimageoffluorescentsignalintensityfromhybridizationtooligonucleotidemicroarraysofuntreatedandsaponin2treatedlabeledwithCy3andCy5.Humanumbilicalveinendothelialcells(Ecv2304)weregrowninDMEMsupplementedwith10%calfbovineseruminanatmosphereof5%CO2.Atabout80%confluence,themediumwasreplacedandthecellswereincubatedinfreshmediumcontaining80mgL-1saponinsfor8hoursat37.IsolationoftotalRNAwasachievedbyusingTRIzolReagent.ThecDNAsderivedfromuntreated(labelledwithCy3)andsaponins2treated(labelledwithCy5)HUVECtotalRNAwerehybridizedtoanarray.Thetwochannelswerepseudocoloredaccordingtothefluorescenceintensity4Cy3Cy5,Cy3Cy5210015,80mgL-1,(AGT)2A(ADRA2R)21(ECE2I)(1)Table1TranscriptionaleffectsofsaponinongenesinHUVECGeneGenebankaccessionNo.FluorescentratioAngiotensinogenK022150136Adenosine2AreceptorXM20098820152Endothelin2convertingenzyme1Z353070132Note:Dataareexpressedasmeans,,,[14],,[15]AGT,ACEAT1,ACEAngII[16],,2A,,,[17,18],,,2A(ADRA2R),,,ADRA2R,ADRA2R,,ADRA2R,,,,,[19],994:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.References:[1]DongJX,HanGY.StudiesontheactiveconstituentsofAnemarrhenaasphodeloides[J].ActaPharmSin(),1992,27(1):26-32.[2]MaBP,DongJX,WangBJ,etal.StudiesonthefurostanolsaponinsfromAnemarrhenaasphodeloides[J].ActaPharmSin(),1996,31(4):271-277.[3]NkashimaN,KimuraI,KimuraM.Isolationofpseudoprototimo2saponinAIIIfromrhizomesofAnemarrhenaasphodeloidesanditshypoglycemicactivityinstreptozotocin2induceddiabeticmice[J].JNatProd,1993,56(3):345-350.[4]WatanabeC,WolfframS,AderP,etal.Theinvivoneuromodulatoryeffectsoftheherbalmed