应用snppcr定量分析造血干细胞移植后nk细胞嵌合体

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1、山西医科大学硕士学位论文QuantitativeanalysisofNKcellchimerismafterhematopoieticstemcelltransplantationbySNP-PCRAbstractObjective:Toinvestigatethefeasibilityandefficiencyofthefluo-rescence-activatedcellsorting(FACS)skill,whichisusedtosortthenaturalkiller(NK)cellsfromtheperipheralbloodofthepatientsafterallogenei

2、chematopoieticstemcelltransplantation(allo-HSCT).Thentoestablishanovelmethod,whichisbasedonsinglenucleotidepolymorphism(SNP)genotypingandcarriedoutbyreal-timepolymerasechainreaction(RT-PCR)withCyclingprobe,forquantitativedetectionofchimerismoftheNKcellsinthepatientsafterallo-HSCT.Theaimsofthesedete

3、ctionsaretoexploretheirapplicationvalue,accuracyandsuperiority,andtoobservetherelationshipsintheearlyperiodbetweentheformationofthischimerismandtheoutcomeofdisease.Methods:7SNPlociswerescreenedfromfivedifferentchromosomesandcorrespondingCyclingprobesandprimerswerecomposedtoidentifyinformativemarker

4、sfordetectingchimerismineachdonor/recipientpairbeforeallo-HSCT.TheNKcellsweresortedfromtheperipheralbloodof20normalpersons(9males,11females)and4donor/recipientpairsbyFACS.DNAofNKcellssampleswasextracted,thenthechimerismrateofeachinformativemarkerwasanalyzedbyCycleavePCRbasedSNP.Theaccuracyandsensit

5、ivityofthisnewmethodwereverifiedbyaseriesofmodelingchimerismfromnormalpersons.Results:①ThepercentageofnormalpersonsNKcellsis(12.86±3.62)%.Aftercellsorting,thepurityis(96.15±2.03)%,andtherecoveryrateis(95.08±2.16)%.ThepercentageofNKcellsofpatientsafterallo-HSCTis(11.01±2.08)%.Aftersorting,thepurityi

6、s(96.22±2.16)%,andtherecoveryrateis(95.27±1.18)%.②Atleastoneinformativemarkercouldbefoundinover78.5%of4donor/recipientpairsand10artificialdonor/recipientpairsformed20normalpersons.③Theslopeofthe7timeamplicationsoftheplasmidwas-2.3681,theinterceptwas36.5919,correlationcoefficientsweremorethan0.995,w

7、hichwasclosetothetheoreticallevel.④ValuesoftheartificialmixedchimerismmeasuredbySNP-PCRareclosetothepredeterminedvalues(P＞0.01).Alinearcorrelationwithartificialchimerismisover0.99,andasensitivityof0.01%prov