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简介：Thepurposeofthestudyistoestablishafluorescencequantitativereversetranscriptionpoly-merasechainresponse(FQ-RT-PCR)methodforthequantitativedeterminationofIL-2mRNAandIL-4mRNAinThcells,withwhichtheThcellsstatusofthepatientswithgynaecologicaltumorsandchronicrenalfailure(CRF)canbeanalyzed.IL-2cDNAandIL-4cDNAwereprepared,andtheplasmidpMD18carryingIL-2cDNAorIL-4cDNAfragmentwasconstructedandclonedasthetemplateforquantitativedetermination.Theprimersandprobeslabelledwith6-carboxy-fluorescein(FAM)and6-carboxy-tetrarnethylrhodamine(TAMRA)wereprepared,andtheexperimentalconditionswereoptimizedtosetuptheFQ-RT-PCRmethodforquantitativedeterminationofIL-2mRNAandEL-4mRNA.Thcellsenrichedfromperipheralbloodmononuclearcells(PBMCs)of20healthyvolunteers(HVs),16gynaecologicalbenign(GB)cases,18gynaecologicalmalignant(GM)tumorcasesand16chronicrenalfailure(CRF)patientsweretestedforIL-2mRNAandIL-4mRNAbyFQ-RT-PCR.Thehouse-keepinggeneβ-actinwasusedastheinternalcontrolgeneoftheexperiment.Thestandardcurveforlogconcentrationofseriesofquantitativetemplatesvsthresholdcycle(CT)wasestablishedbylinearregression,andthelinearrangewas102-107copies/μl.TheimprecisiontestshowedtheCVofinter-assayandintra-assayofahighcontentsamplebyFQ-RT-PCRwere7.8%and12.5%,respectively.TheCVofinter-assayandintra-assayofalowcontentsamplewere10.8%and19.5%,respectively.TheIL-2mRNAexpressionsinThofthepatientswithgynaecologicalmalignanttumor(comparedwiththeHVsandthepatientswithgynaecologicalbenigndisease)andinThoftheCRFpatients(comparedwiththeHVs)weredeclinedsignificantlyandatthesametimetheIL-4mRNAexpressionincreasedsignificantly(P<0.001).Asimple,sensitiveandaccurateFQ-RT-PCRmethodforthequantitativedetectionofIL-2mRNAandIL-4mRNAhasbeenestablished.TheIL-2mRNAandIL-4m

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简介：Toinvestigatetheroleofnegative-regulatoryfactorsA20,IRF-4andTRAF4ofthetoll-likereceptor(TLR)signalpathwaysinimmunologicalpathogenesisofKawasakidisease(KD),48childrenwithKawasakidisease,16childrenwithinfectiousdisease(ID)and16age-matchedhealthychildrenwerestudied.Reverse-transcriptionPCR(RT-PCR)andreal-timePCRwereusedtoevaluatetheexpres-sionlevelsofnegative-regulatoryandeffectivefactorsintoll-likereceptor4(TLR4)signalpathwaysandproinflammatoryfactorsinperipheralbloodmonocyte/macrophage(MC).Inthisstudy,expressionlevelsofTLR4,MD-2,MyD88,IRAK-4,TRAF6,TAK1,andTAB2mRNAinKDgroupweredetectedtobeelevatedsignificantlyduringacutephaseofKD.Transcriptionlevelsofnegative-regulatoryfactorsA20,IRF-4andTRAF4mRNAinKDorIDpatientsincreasedremarkably.However,expressionsofIRF-4andTRAF4inKDpatientsweredetectedtobelowerthanthatinIDpatients,exceptthattran-scriptionlevelsofA20werefoundtobehigherthanthatinIDpatients.Simultaneously,expressionsofproinflammatorycytokinessuchasL-1β,IL-6andTNF-αinKDpatientsweresignificantlyelevatedcom-paredwiththoseinIDpatients.Furthermore,itwasfoundthatstimulationoflipopolysaccharide(LPS)remarkablyup-regulatedtheexpressionsofnegative-regulatoryfactorsA20,IRF-4andTRAF4inKDpa-tientsorhealthyvolunteers.ThemRNAlevelsofallthethreefactorsinKDpatientswerefoundtobelowerthanthatinthelatter.Inaddition,transcriptionlevelsofIRF-4andTRAF4inKDpatientswithcoronaryarterylesion(KD-CAL~+)weredetectedtobelowerthanthoseinKDpatientswithoutcoronaryarterylesion(KD-CAL~-)duringacutephase,whilethatofA20inKD-CAL~+groupwerelowerthanthatinthelatter.AndthelevelsofexpressionsofproinflammatorycytokinesinKD-CAL~+groupwerefoundtobehigherthanthoseinKD-CAL-group(P<0.01).Thesefindingssuggestthataberrantexpressionofnegative-regulatoryfactorsofTLRssignalpathwaysmaybeinvolved

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