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1、短發(fā)夾RNA靶向抑制EGFR基因?qū)θ寺殉舶㏒kov：張紅玲陳愛平楊蕊蕊【摘要】目的探討RNA干擾（RNAi）對卵巢癌細胞表皮生長因子受體（EGFR）基因表達的影響及其效應。方法體外合成EGFR的DNA模板序列和pSilencer2.1-U6neo質(zhì)粒構(gòu)建編碼shRNA的表達載體，應用脂質(zhì)體Lipofectamine2000將其轉(zhuǎn)染到人卵巢癌Skov-3細胞，采用半定量RT-PCR、免疫細胞化學技術(shù)檢測轉(zhuǎn)染前后Skov-3細胞EGFR基因的變化；采用AnnexinV/PI雙染色標記的流式細胞儀檢測siRNA誘導的細胞凋亡，PI染色檢測細胞周期阻滯。結(jié)果成功構(gòu)建pSilence
2、r-EGFR短發(fā)卡狀siRNA真核表達載體；靶向EGFR的序列特異性的siRNA可以有效抑制Skov-3細胞EGFR基因的表達；流式細胞分析顯示，Skov-3細胞凋亡率明顯增加，細胞周期出現(xiàn)明顯的G0/G1期阻滯。結(jié)論RNAi沉默EGFR表達可以誘導卵巢癌Skov-3細胞凋亡，并使卵巢癌Skov-3細胞停滯在G0/G1期，RNAi可作為研究卵巢癌發(fā)生發(fā)展機制和基因治療的有效工具。【關鍵詞】卵巢腫瘤；受體,表皮生長因子；RNA干擾；細胞凋亡ABSTRACT］ObjectiveToexploretheeffectofRNAinterference（RNAi）-mediatedi
3、nhibitiononepidermalgroidvectortoconstructtheshRNAexpressionvector.AftertheconstructedvectoranovariancancerSkov-3cellsusinglipofectamine2000,semi-quantitativeRT-PCRandimmunocytochemistryassayetricanalysisindicatedthattheapoptosisrateofSkov-3cellsincreasedsignificantlyandanobviouscellcyclea
4、rrestatG0/G1phaseediatedsilencingofEGFRgeneexpressioncaninducecellapoptosisandcellcyclearrestatG0/G1phase.Thus,RNAicanbeusedasaneffectivetooltoexplorethemechanismunderlyingthedevelopmentandprogressionofovariancancerass;Receptor,epidermalgroahasahighermortalityrateamonggynecologicalmalignan
5、cies［1］.Over-expressionofEGFR,asinmostcasesofovariancarcinoma,isassociatedalignancyandprognosis［2］.EGFRhasrecentlyreceivedconsiderableattentioninovariancancerresearch,sincethis170000glycosylatedmembrane-spanningproteinreceptorisover-expressedupto75%inprimarycases［3］.Furthermore,activationo
6、fEGFRsignalingpathetastasisanddecreasedapoptosis.EGFRisthusconsideredasacriticaltargetinthetreatmentofthiscancer［4］.RNAinterference（RNAi）,alsoknoepost-transcriptionalcontrolmechanismsviaRNAisdegraded［5-7］.Forthisreason,RNAitechnologyanovariancancerSkov-3celllinetheDepartmentofPathogenicMic
7、robiologyofQingdaoUniversity.EukaryoticexpressionplasmidpSilencer2.1-U6neoAmbion（USA）.Lipofectamine2000Invitrogen（USA）.PureYieldPlasmidMidiprepSystemega.Propidiumiodide（PI）andribonucleasea.AnnexinV-FITC/PIeiBioengineeringpany.Anti-EGFRantibodybion’sonlinesofta
