【摘要】 通过RNA干扰实验沉默酪蛋白激酶1α(Casein Kinase1α,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夹表达载体pGenesil-CK1α-S1、pGenesil-CK1α-S2、pGen-esil-CK1α-S3和一个无义错配载体pGenesil-NK,分别转染A549细胞,用G418筛选出阳性克隆,得到A549-S1、A549-S2、A549-S3及A549-NK细胞株.再通过RT-PCR和W estern b lotting方法,分别检测了CK1α基因的mRNA和蛋白的表达情况.比对A549-NK,A549-S1、A549-S2及A549-S3 mRNA水平的抑制率分别为84.3%、57.9%和61.8%(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A549-S1细胞进行后续实验.细胞计数绘制生长曲线、CCK-8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,A549-S1与A549和A549-NK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);CCK-8抑制率为(26.2±1.5)%(P<0.05);S期减少(75.0±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.更多还原

【Abstract】 Casein Kinase 1α(CK1α) is silenced by RNA interference for investigating the effect on A549 cells’ growth and proliferation.First,three shRNA plasmids specifically targeting different sites of CK1α(pGenesil-CK1α-S1,pGenesil-CK1α-S2,pGenesil-CK1α-S3) and one nonsense shRNA plasmid(pGenesil-NK) are constructed and transfected into A549 cells,and the stable transfected cell lines(A549-S1,A549-S2,A549-S3 and A549-NK) are sorted out by G418.Secondly,the expression of mRNA and protein of CK1α is detected by semi-quantitative RT-PCR and western blotting.Compared with pGenesil-NK,expression of A549-S1,A549-S2 and A549-S3 are dereased by 84.3%,57.9% and 61.8% respectively at mRNA level(P<0.001);78.4%,51.3% and 59.9% respectively at protein level(P<0.001).Thus A549-S1 is chosen to do further experiment.Thirdly,cell growth curve is drawn by cell counting;cell proliferation is measured by CCK-8 and cell cycle distribution is assessed by flow cytometry for investigating the difference among A549,A549-NK and A549-S1 cells’ growth and proliferation.Experimental results show that the A549-S1 cells’ IR is(37.7±2.2)% in cell counting experiment(P<0.01) and(26.2 ± 1.5)% in CCK-8 assay(P<0.05) respectively,and the S and G2/M phase in A549-S1 cells are significantly reduced by(75.0 ± 4.0)%(P<0.01) and(45.2±5.4)%(P<0.05) compared with the average values of A549 and A549-NK.Therefore,silencing of CK1α gene can inhibit the growth and proliferation of A549 cells efficiently.更多还原