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《實(shí)時(shí)熒光定量rt-pcr檢測人外周血單個(gè)核細(xì)胞中foxp3 mrna的表達(dá)》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、實(shí)時(shí)熒光定量RT-PCR檢測人外周血單個(gè)核細(xì)胞中FOXP3mRNA的表達(dá)作者：蔣衛(wèi)平，丁茂文，朱亞非，李冰，朱晚林，李欣華，林菲菲【摘要】目的：建立實(shí)時(shí)熒光定量逆轉(zhuǎn)錄-多聚酶鏈反應(yīng)(real-timefluorescencequantitativeRT-PCR)檢測人外周血單個(gè)核細(xì)胞中FOXP3mRNA的方法，并研究其與CD4+CD25+Treg細(xì)胞活性相關(guān)性。方法：提取人外周血單個(gè)核細(xì)胞總RNA，將mRNA逆轉(zhuǎn)錄成cDNA，以β-actin為內(nèi)參照，實(shí)時(shí)熒光定量RT-PCR檢測29例哮喘患兒及24例同齡對(duì)照F
2、OXP3mRNA的相對(duì)表達(dá)量，采用融解曲線和瓊脂糖電泳鑒定PCR產(chǎn)物特異性；同時(shí)采用流式細(xì)胞技術(shù)檢測CD4+CD25+Treg細(xì)胞含量，分析兩者相關(guān)性。結(jié)果：哮喘患兒組CD4+CD25+Treg細(xì)胞百分率明顯低于同齡對(duì)照組，P＜0.01；FOXP3mRNA的表達(dá)也顯著降低，P＜0.05；FOXP3和β-actin的融解曲線分析表明均僅有單一峰，Tm值分別為82.4℃和87.8℃；瓊脂糖電泳顯示都僅有單一擴(kuò)增產(chǎn)物。結(jié)論：應(yīng)用SYBRGreenⅠ實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測FOXP3mRNA表達(dá)水平簡便易行、結(jié)
3、果穩(wěn)定可靠。初步結(jié)果證實(shí)，外周血CD4+CD25+Treg細(xì)胞數(shù)量和FOXP3mRNA表達(dá)有相同的趨勢，存在一定的相關(guān)性?！娟P(guān)鍵詞】12實(shí)時(shí)熒光定量RT-PCR；哮喘；FOXP3；CD4+CD25+調(diào)節(jié)性T細(xì)胞 Abstract:Objective:Toestablishareal-timefluorescentquantitativereversetranscriptionpolymerasechainreaction（RT-PCR）todetectFOXP3mRNAexpressioninhumanper
4、ipheralbloodmononuclearcells（PBMCs）andtoinvestigatethecorrelationsbetweenactivityofCD4+CD25+regulatoryTcellsandexpressionofFOXP3mRNA.Methods:TotalRNAwasextractedwithTrizolfromhumanPBMCsandmRNAwastranscribedreverselyintocDNA.Real-timefluorescentquantitativeRT
5、-PCRwithβ-actinastheinternalcontrolgenewasusedtodetecttheexpressionlevelsofFOXP3mRNAin29pediatricpatientswithasthmaand24age-matchedhealthychildren.ThespecificityofPCRproductionswasidentifiedwiththedissociationcurvesandagarosegelelectrophoresis.Flowcytometrya
6、nalysiswasusedtoassessthepercentageofCD4+CD25+regulatoryTcells.Thecorrelationbetweentheexpressionandactivitywasanalyzed.Results:ThepercentagesofCD4+CD25+regulatoryTcellsingroupofasthmaweresignificantlylowerthanthoseingroupofcontrol(P<0.01)，sodidtheexpress
7、ionsofFOXP3mRNA(P<0.05).TheanalysisofdissociationcurvesonFOXP3andβ-actinshowedthatbothofthemhadonlyonesimplespikeand12theTmvalueswere82.4℃and87.8℃，respectively.TheagarosegelelectrophoresisofthemshowedonlysinglePCRproducteach.Conclusion:Itisaneasyandreliab
8、letesttodetecttheexpressionlevelofFOXP3mRNAbySYBRGreenⅠreal-timefluorescencequantitativeRT-PCRandthepreliminaryexperimentalresultshowsthatthepercentagesofCD4+CD25+regulatoryTcellsandtheexpressio
