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《靶向CK1α 基因SHRNA 表達(dá)載體的構(gòu)建及其對A549 細(xì)胞增殖的影響》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫。
1、2010年3月湖南師范大學(xué)自然科學(xué)學(xué)報(bào)Vol.33No.1第33卷第1期JournalofNaturalScienceofHunanNormalUniversityMar.,2009靶向CK1α基因shRNA表達(dá)載體的構(gòu)建及其對A549細(xì)胞增殖的影響3鄒飛雁,姜少杰,劉曉,李俐娟,劉婉君(暨南大學(xué)生殖免疫研究所,中國廣州510632)摘要通過RNA干擾實(shí)驗(yàn)沉默酪蛋白激酶1α(CaseinKinase1α,CK1α)基因,探討其對肺癌細(xì)胞A549生長增殖的影響.首先構(gòu)建3個(gè)針對CK1α基因不同位點(diǎn)的短發(fā)
2、夾表達(dá)載體pGenesil2CK1α2S1、pGenesil2CK1α2S2、pGen2esil2CK1α2S3和一個(gè)無義錯(cuò)配載體pGenesil2NK,分別轉(zhuǎn)染A549細(xì)胞,用G418篩選出陽性克隆,得到A5492S1、A5492S2、A5492S3及A5492NK細(xì)胞株.再通過RT2PCR和Westernblotting方法,分別檢測了CK1α基因的mRNA和蛋白的表達(dá)情況.比對A5492NK,A5492S1、A5492S2及A5492S3mRNA水平的抑制率分別為84.3%、57.9%和6118
3、%(P<0.001);蛋白質(zhì)水平的抑制率分別為78.4%、51.3%和59.9%(P<0.001).選擇干擾效率較好的A5492S1細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn).細(xì)胞計(jì)數(shù)繪制生長曲線、CCK28法觀察細(xì)胞增殖狀況和流式細(xì)胞儀檢測細(xì)胞所處周期時(shí)相來分析轉(zhuǎn)染CK1α基因shRNA表達(dá)載體對A549細(xì)胞生長增殖的影響.實(shí)驗(yàn)結(jié)果顯示,A5492S1與A549和A5492NK兩對照組的平均值比較,細(xì)胞計(jì)數(shù)抑制率為(37.7±2.2)%(P<0.01);CCK28抑制率為(26.2±1.5)%(P<0105);S期減少(75
4、.0±4.0)%(P<0.01);G2/M期減少(45.2±5.4)%(P<0.05).因此,轉(zhuǎn)染CK1α基因shRNA表達(dá)載體可導(dǎo)致CK1α基因的沉默,明顯抑制A549細(xì)胞的生長和增殖.關(guān)鍵詞酪蛋白激酶1α;A549;RNA干擾;細(xì)胞增殖中圖分類號R73412文獻(xiàn)標(biāo)識(shí)碼A文章編號100022537(2010)0120101207ConstructionofanExpressionVectorCarryingShortHairpinRNATargetingCK1αGeneandItsEffectson
5、A549CellspGrowthZOUFei2yan,JIANGShao2jie,LIUXiao,LILi2juan,LIUWan2jun(InstituteofproductiveImmunology,JinanUniversity,Guangzhou510632,China)AbstractCaseinKinase1α(CK1α)issilencedbyRNAinterferenceforinvestigatingtheeffectonA549cellspgrowthandproliferatio
6、n.First,threeshRNAplasmidsspecificallytargetingdifferentsitesofCK1α(pGene2sil2CK1α2S1,pGenesil2CK1α2S2,pGenesil2CK1α2S3)andonenonsenseshRNAplasmid(pGenesil2NK)areconstructedandtransfectedintoA549cells,andthestabletransfectedcelllines(A5492S1,A5492S2,A54
7、92S3andA5492NK)aresortedoutbyG418.Secondly,theexpressionofmRNAandproteinofCK1αisdetectedbysemi2quantitativeRT2PCRandwesternblotting.ComparedwithpGenesil2NK,expressionofA5492S1,A5492S2andA5492S3aredereasedby84.3%,57.9%and61.8%respectivelyatmRNAlevel(P<0.
8、001);78.4%,51.3%and59.9%respectivelyatproteinlevel(P<0.001).ThusA5492S1ischosentodofurtherexperiment.Thirdly,cellgrowthcurveisdrawnbycellcounting;cellproliferationismeasuredbyCCK28andcellcycledistri2butionisassessedbyflowcytometr
