头发中抽提DNA.ppt

头发中抽提DNA并用PCR RFLP法鉴定CYP2C9基因型1 感受科学研究2 接触科学研究3 自己动手做科学研究 由简单到复杂 本科生 硕士生 博士生学习 研究 创新新发现 新方法 新结论 Homework mgkosff2005 5 8 Index Part1reviewoftwopapersPart2discussionofmyown emphasis Part3someinformationPart4supplement Part1Reviewoftwopapers IdentificationofanovelvariantCYP2C9alleleinChinese Part1Reviewoftwopapers Backgroundinformation CytochromeP4502C9 CYP2C9 amemberoftheCYP2Cenzymesubfamily ranksamongthemostimportantdrugmetabolizingenzymesinhumans Geneticpolymorphism SNP ofCYP2C9 wild typeproteinCYP2C9 1CYP2C9 2 Arg144Cys CYP2C9 3 Ile359 Leu CYP2C9 4 Ile359Thr CYP2C9 5 Asp360Glu CYP2C9 6 nullallele and5 noncodingregionTolbutamideiswidelyacceptedasaprobesubstratefortheassessmentofhepaticCYP2C9activity Part1Reviewoftwopapers Methodsandresults Tolbutamidepharmacokinetics Theproceduresisomitted ThePMstudysubject splasmahalf lifeisabout9 15timesthatoftheCYP2C9 1 1orCYP2C9 1 3carriersandisevenslowerthantheCYP2C9 3 3genotypesubjects SequencingofCYP2C9 CYP2C9genewasamplifiedbyPCR andGenotypingtests PCR RFLPanalysis RevealingthestudysubjectisheterozygousforCYP2C9 3andapreviouslyunreportedmutationinexon2 ThismutationassociateswithaT269CtransversionoftheCYP2C9genethatleadstoaLeu90Prosubstitutionintheencodedprotein Part1Reviewoftwopapers Frequencyanalysis PCR RFLPanalysis TheT269CchangeinthesequenceCTTGGcreatesanewrecognitionsiteofEcoRII CCTGG suchthatEcoRIIdigestedthePCRproductsoftheCYP2C9T269Calleleintofragmentsof192bp 195bpand302bp whereasthePCRproductsoftheCYP2C9 1alleleweredigestedintofragmentsof387bpand302bp Thesedatacorrespondtoanallelefrequencyof1 02 95 confidencelimitsof0 2to3 0 intheChinesepopulation Part1Reviewoftwopapers ConclusionThisstudysuggeststhatthisnovelCYP2C9allelewascorrelatedwithreducedplasmaclearanceofdrugsthataresubstratesforCYP2C9 Part1Reviewoftwopapers ROLEOFCYP2C9ANDITSVARIANTS CYP2C9 3ANDCYP2C9 13 INTHEMETABOLISMOFLORNOXICAMINHUMANS Part1Reviewoftwopapers Backgroundinformation CYP2C9isanimportantmemberofthecytochromeP450enzymesuperfamilywithsome12CYP2C9alleles 1 12 beingpreviouslyreported Recently weidentifiedanewCYP2C9allelewithaLeu90PromutationinaChinesepoormetabolizeroflornoxicam Othersarethesameasabove Part1Reviewoftwopapers Methodsandresults QuantificationofCYP2C9Protein WesternBlotting AllconstructsyieldedimmunodetectableCYP2C9protein asdidhumanlivermicrosomes TheexpressedproteinlevelofCYP2C9 1was7 51pmol mgS9protein andtheexpressionlevelsofvariantCYP2C9 3and 13were69 9 and35 5 respectively ofthatofCYP2C9 1 InVitroLornoxicam5 HydroxylationofRecombinantCYP2C9Protein TransfectionofCOS 7CellsandPreparationofPostmitochondrialSupernatant S9 Thenlornoxicam5 hydroxylationactivityoftherecombinantCYP2C9proteinwasdeterminedbyHPLC BothCYP2C9 3andCYP2C9 13exhibitedlowerintrinsicclearanceoflornoxicam P 0 01 thandidwild typeCYP2C9 1 Clearly theactivityofCYP2C9 13islowerthanthatofCYP2C9 3 Part1Reviewoftwopapers InVivoLornoxicamMetabolism Inbrief afterasingleoraldoseof8mgoflornoxicam bloodsampleswerecollectedbeforedosing andat0 5 1 1 5 2 2 5 3 4 6 8 10 13 and24hpostdose Plasmaconcentrationsoflornoxicamweredeterminedusingavalidatedliquidchromatography tandemmassspectrometrymethod TheAUCoflornoxicamincreased1 9 foldandCL Fwasdecreasedby44 inCYP2C9 1 13individualscomparedwithCYP2C9 1 1individuals SimilarchangeswerefoundinCYP2C9 1 3individuals Part1Reviewoftwopapers Conclusion TheLeu90ProsubstitutionofCYP2C9 13markedlydecreasestheintrinsicclearanceoflornoxicaminvitroandinvivo ThereductioninactivityduetoCYP2C9 13isgreaterthanthatduetoCYP2C9 3invitro Part2discussionofmyown First howthisSNPcandisruptedtheactivitysomuchisaproblemabouttherelationshipbetweenstructureandfunctionofaprotein Maybeaeffectofthegenelevelisinvolved whichismoredifficulttounderstand Asissaidinthatpaper theLeuresidueliesinaloop farfromthehemebindingsites andisnottheplacewheretwosubunitsinteractwitheachother Someinformation 7twochainsandchainA Soitispuzzledwhytheactivitydecreasessomuch Fromthecrystalstructure Ihavefoundtwointerestingthings Part2discussionofmyown OneisthattheLeuresidueshouldbeapartofaalphahelix fromthesecondarystructurepredictionmentionedbelowandtheagreementofhydrophobicityoftheLeuandneighboringresiduestoformalongeralphahelixthanthecrystalstructure Butwhatdisturbthehelixandforcethemtobecomelessenloopform maybeitistheneighboringloopandabetasheetnearitthatconstructthisform Someinformation 7chainAandLocalresiduesnearLeu IthinkthattheLeuresidueliesbetweenthosetwosecondarystructuresanditisforcedtoloosetoaloopinsteadofhelixstructure AndtheSNPthatProreplacesLeupossiblymeansthatthesemirigidloopbecomesmorerigidloopasProismorerigidthanLeu whichhaveaneffectontheneighboringloopandthebetasheet Part2discussionofmyown Andtheneighboringloopseemstocontroltheaccessibilityofdrugstoheme whichinresultcausestheactivitytodecrease TheprocessindetailmaybethattheProterminatesthealphahelix andmakesaturnerwhichmakesthenextbackbonebendtooneside andthusthebendingbackbonepushthenearloopandthatloopismoretightthanbefore Thenthatmoretightloopcloseorbarrierthedoorthroughwhichthedrugscantouchtheheme ItisthewholereactionIsupposed Part2discussionofmyown Residue81 95eavkealidldeefsThebluerepresentsthehydrophilicresidue Itisobviouslythatthe dldee hasthesamehydrophilicpatternas eavke whichformsahelix Part2discussionofmyown ThehelixandtheloopinthecenteraretheresiduesneartheLeu90 Anotherloopandasheetencircleit Part2discussionofmyown Theyellowpartsistheloopwhichfunctionsasagate Iguess Inthecenterofthesubunitisaheme Part2discussionofmyown TheyellowrepresentstheLeuresidue ThearrowpointsthemovementwhentheLeu90isreplacedwithPro Iguess Part2discussionofmyown AnotheristhattheLeuisneartheconnectionoftwodomainsofonesubunit Isupposethatthechangeintherigidityoftheloopcanbringaseriesofeffectonthetwodomains whichbothencircletheheme thelargedomainismoreimportantinregulatetheaccessibilityofthehemeanddrugs theexactprocessisnotconsideredyet Part2discussionofmyown Second thepapermentionedthatthefrequencyanalysiswascarriedoutamong147unrelatedHanChinesemales Ithinkthesampleisnotsoreliabletosupporttheconclusionthattheallelefrequencyof1 02 95 confidencelimitsof0 2to3 0 intheChinesepopulation Thereasonisveryapparent Chinahasapopulationof1 3billion andconsistsofmorethanfiftypeoples Part2discussionofmyown Andthesampleonlyconsistsofmales notasinglewoman Thetotalnumber thesexandpeopleshouldincludelargertoanextentthatmorereliable characteristicdatacanbeobtained SofurtherinvestigationneedstobemadetoconcludeamoreaccuratefrequencyamongtheChinesepeople Part2discussionofmyown Third thoughitisanunfortunateforthecarrierwhocarriesbothallelesthataremutant heterozygousforCYP2C9 3andapreviouslyunreportedmutation itisquiteluckyfortheresearcherwhowanttoknowthatwhetherthepreviouslyunreportedmutationistwomutationsoccurredonthesameallele BecausetheresultshowsthattheparentsareheterozygousforthenewCYP2C9T269C 1alleleandCYP2C9 1 3 respectively ThedeductioncanbemadethatthesubjectobtainedthenewCYP2C9T269CmutantalleleandCYP2C9 1fromeitherparentrespectively ThefinalconclusionisthatthenovelmutationandtheCYP2C9 3mutationoccuronseparatealleles Part2discussionofmyown ButsupposedthateitherparenthasbothCYP2C9T269CandCYP2C9 3asthesubjectandtheotherparenthasCYP2C9T269C thenwhattheconclusionwillbe Itisobviouslythattheresultwillbeconfused Onepossibilityisthatthesubjectderivedanallelethathasbothmutation andanotherallelefromanotherparent Analternationisthesameasthepapermade SoIthinkthemethodofanalyzingtheparentsgenotypetoconfirmwhetherthepreviouslyunreportedmutationistwomutationsoccurredonthesamealleleisnotaquitecommonone andIproposethemethodtodetectpresenceofthewild typeCYP2C9proteinisacommonone Part2discussionofmyown AndtheFourth thearticlementionedthattheproteinlevelsofcDNA expressedCYP2C9 3and 13inpostmitochondrialsupernatant S9 fromCOS 7cellswerelowerthanthoseinwild typeCYP2C9 1 Andthatlowertranscription translationefficiency proteinstability theimmunoreactivityofmutantCYP2C9sarepresentedaspossiblereasons Frommypointofview Ithinkthatconsideringitisanone pointmutationandnotanycontrolsequenceischanged itisunreasonabletotakethelowertranscriptionortranscriptionefficiencyasthereasons Sothelessproteinstabilitymaybemoreconceivable becauseitisreasonabletodegradelessefficientproteinbytheubiquitinpathway thoughtheexactmechanismisunknown TheLeu90isonthesurfaceoftheproteinasillustratedbythecrystalstructure sothelessimmunoreactivitymayalsocontributetothatresult Part2discussionofmyown AndIsuspectthattheLeu90Propointmutationmayhaveaeffectonthegenelevel thatistosaythatmaybeanewsiteforintronsplicingiscreated sotheaminoacidsequenceofthatexonpossiblybechanged Ifitistrue lowertranscription translationefficiency proteinstability theimmunoreactivityallshouldbetakenintoconsideration ThusitmayexplainwhysomuchactivityisdecreasedwhenitisonlyanSNPwhichisnottheheme bindingregionandfarfromthebindingpocketofsubstrate Furtherexperimentsshouldbedonetoexplorethemajorreasonsforthat Part2discussionofmyown Whatismore thearticleonlyobtainedonedrugpharmacokinetics Maybeitisanexception Iftheexacteffectsonthekineticsofotherdrugsneedtobeknown everydrugshouldbetestedforsure Somoreexperimentsofthepharmacokineticsofdifferentdrugsshouldbeonschedule Part2discussionofmyown Last itbringsanotherproblemthatindividualswithCYP2C9 1 3andCYP2C9 1 13genotypesrevealthesameextentofreductioninoralclearanceoflornoxicamdespitethefactthat invitro CYP2C9 13isassociatedwithalowerintrinsicclearanceoflornoxicamthanisCYP2C9 3 OnereasonIsupposeisthattheresultshowsthattheproteinlevelofCYP2C9 3ishigherthanCYP2C9 13 sotheinfluenceonthephenotypeofdecreasedactivityofCYP2C9 3isbiggerthanthatofCYP2C9 13 Part2discussionofmyown AnotherreasonmaybethatsomeotherproteinsinvivomayhelpCYP2C9 13 butnosuchproteinscanhelpCYP2C9 3 Sothetotaleffectinvivoisfundamentallythesamebetweenthem Andtheproteinismadeupoftwohomosubunits andtheinfluenceofonemutantsubunitonthetotalactivitymaybedifferent Part3Someinformation HomosapienscytochromeP450 family2 subfamilyC polypeptide9 mRNA cDNAcloneIMAGE 4722940 completecds 241gaagcagtgaaggaagccctgattgatctt cggagaggagttttctggaagaggcattttc Part3Someinformation ThegeneislocatedwithinaclusterofcytochromeP450genesonchromosome10q24 Thisproteinlocalizestotheendoplasmicreticulumanditsexpressionisinducedbyrifampin exon2 ctctcaaaggtctatggccctgtgttcactctgtattttggcctgaaacccatagtggtgctgcatggatatgaagcagtgaaggaagccctgattgatctt cggagaggagttttctggaagaggcattttcccactggctgaaagagctaacagaggatttg Part3Someinformation Chain1OG2 ACompoundCytochromeP4502C9TypePr