血清htertmrna實(shí)時熒光pcr定量方法的建立及初步應(yīng)用

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1、血清hTERTmRNA實(shí)時熒光PCR定量方法的建立及初步應(yīng)用劉陽[通信作者]劉陽，Email:liuyang081@gmail.com，電話：（0731）88639075，胡梅2，吳婭娜3喻紅波1（410006長沙，1武警湖南總隊(duì)醫(yī)院檢驗(yàn)科2湖南中醫(yī)藥大學(xué)生物化學(xué)教研室3湖南中醫(yī)藥大學(xué)圖書館）[摘要]目的建立血清人端粒酶逆轉(zhuǎn)錄酶（humantelomerasereversetranscriptase,hTERT）mRNA實(shí)時熒光定量PCR檢測方法，并初步應(yīng)用于胃癌檢測。方法以hTERT基因?yàn)榘谢蛟O(shè)計

2、引物，構(gòu)建攜帶hTERTcDNA的質(zhì)粒pMD-hTERT，建立血清hTERTmRNA實(shí)時熒光PCR定量方法，檢測48例胃癌患者，58例健康人血清端粒酶逆轉(zhuǎn)錄mRNA表達(dá)水平并分析其差異。結(jié)果建立血清hTERTmRNA表達(dá)的實(shí)時熒光定量PCR方法，胃癌患者h(yuǎn)TERTmRNA相對表達(dá)水平顯著高于健康人（p<0.01），與腫瘤大小和分期、分化等級明顯相關(guān)（p<0.01），與年齡、性別、以及腫瘤數(shù)量無明顯關(guān)聯(lián)（p＞0.05）。結(jié)論本研究成功建立了血清hTERTmRNA表達(dá)的實(shí)時熒光定量PCR方法，實(shí)驗(yàn)表明檢測

3、血清hTERTmRNA有助于胃癌診斷和研究。[關(guān)鍵詞]人端粒酶逆轉(zhuǎn)錄酶，實(shí)時熒光定量PCR，胃癌[中圖法分類號][文獻(xiàn)標(biāo)識碼]Real-timefluorescencequantitativePCRassayforhumanTelomerasereversetranscriptasemRNAinserumandpreliminaryapplicationinGastriccancerLiuYang1,HuMei,WuYana,YuHongbo(1，4DepartmentofClinicalLabora

4、tory,HunanProvincialCorpsHospital,ChinesePeople’sArmedPoliceForces,2,3HunanUniversityofTraditionalChineseMedicine,Changsha,410006,China)[Abstract]ObjectiveToestablishreal-timefluorescencequantitativepolymerasechainreactionmethodfordetectinghumantelomera

5、sereversetranscriptasemRNAinserum,andstudieditsclinicalsignificanceingastriccancerpatients.MethodsDesignedhTERTprimersandconstructedplasmidpMD-hTERTcarryinghTERTcDNA,andestablishedaquantitativePCRassaytodetecthTERTmRNAexpressionlevelsinserumof48gastricc

6、ancerpatientsand58healthyindividuals.ResultsAreal-timefluorescencequantitativePCRmethodfordetectinghTERTmRNAlevelsinserumwasestablished,TheexpressionlevelofhTERTinserumofgastriccancergroupwassignificantlyhigherthanhealthygroup.Therelativeexpressionlevel

7、ofhTERTmRNAwasdemonstratedtobeindependentlycorrelatedwithtumorsize,gradeandTNMstage,whilenotcorrelatedwithage,genderandnumberoftumors.ConclusionThereal-timequantitativePCRfordetectingserumhTERTmRNAwasestablishedsuccessfullywiththecharacterofhighspecific

8、ity,sensitivityandrepeatability,whichmightprovideanoveldiagnosisandresearchtoolforgastriccancer.[KeyWords]humantelomerasereversetranscriptase，real-timefluorescencequantitativepolymerasechainreaction，Gastriccancer端粒酶在hTERT的作用下延長隨細(xì)