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1、2010年3月湖南师范大学自然科学学报Vol.33No.1第33卷第1期JournalofNaturalScienceofHunanNormalUniversityMar.,2009靶向CK1α基因shRNA表达载体的构建及其对A549细胞增殖的影响3邹飞雁,姜少杰,刘晓,李俐娟,刘婉君(暨南大学生殖免疫研究所,中国广州510632)摘要通过RNA干扰实验沉默酪蛋白激酶1α(CaseinKinase1α,CK1α)基因,探讨其对肺癌细胞A549生长增殖的影响.首先构建3个针对CK1α基因不同位点的短发
2、夹表达载体pGenesil2CK1α2S1、pGenesil2CK1α2S2、pGen2esil2CK1α2S3和一个无义错配载体pGenesil2NK,分别转染A549细胞,用G418筛选出阳性克隆,得到A5492S1、A5492S2、A5492S3及A5492NK细胞株.再通过RT2PCR和Westernblotting方法,分别检测了CK1α基因的mRNA和蛋白的表达情况.比对A5492NK,A5492S1、A5492S2及A5492S3mRNA水平的抑制率分别为84.3%、57.9%和6118
3、%(P<0.001);蛋白质水平的抑制率分别为78.4%、51.3%和59.9%(P<0.001).选择干扰效率较好的A5492S1细胞进行后续实验.细胞计数绘制生长曲线、CCK28法观察细胞增殖状况和流式细胞仪检测细胞所处周期时相来分析转染CK1α基因shRNA表达载体对A549细胞生长增殖的影响.实验结果显示,A5492S1与A549和A5492NK两对照组的平均值比较,细胞计数抑制率为(37.7±2.2)%(P<0.01);CCK28抑制率为(26.2±1.5)%(P<0105);S期减少(75
4、.0±4.0)%(P<0.01);G2/M期减少(45.2±5.4)%(P<0.05).因此,转染CK1α基因shRNA表达载体可导致CK1α基因的沉默,明显抑制A549细胞的生长和增殖.关键词酪蛋白激酶1α;A549;RNA干扰;细胞增殖中图分类号R73412文献标识码A文章编号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
