摘要
目的探讨TPX2基因沉默对人肺腺癌A549细胞增殖的影响。方法构建3个靶向TPX2基因的短发夹RNA(shRNA)表达质粒,转染A549细胞,RT-PCR检测TPX2基因mRNA的转录水平;筛选干扰效果最佳的质粒转染的细胞,RT-PCR检测增殖相关基因CDK4和CyclingD1 mRNA的转录水平,Western blot检测TPX2蛋白的表达水平,显微镜下观察细胞的生长情况,MTT法检测细胞的增殖活力。结果 TPX2 shRNA-3质粒干扰效果最佳,有效沉默TPX2基因后,A549细胞中TPX2基因mRNA的转录水平及蛋白的表达水平均受到明显抑制(P<0.01),与空白对照组比较,抑制率分别为73.8%和82.8%;CDK4和CyclingD1基因mRNA的转录水平也明显降低(P<0.01或<0.05),与空白对照组比较,抑制率分别为76.7%和67.3%;A549细胞的生长和增殖也明显受到抑制,转染后72 h,增殖抑制率可达61.55%(P<0.05)。结论成功构建了靶向TPX2基因的shRNA表达质粒,其在mRNA和蛋白水平有效抑制A549细胞TPX2基因的表达后,细胞生长受到抑制,增殖能力减弱。
Objective To investigate the effect of TPX2 gene silence on proliferation of human lung adenoma A549 cells.Methods Three shRNA plasmids for TPX2 gene were constructed and transfected to A549 cells.The transcription level of TPX2 mRNA was determined by RT-PCR.The cells transfected with the plasmid showing satisfactory interfering effect were screened,and determined for the transcription levels of mRNAs of proliferation-related genes CDK4 and CyclinD1 by RT-PCR,and for expression level of TPX2 protein by Western blot.The growth of cells was observed by microscopy,while the proliferation activity was determined by MTT method.Results TPX2 shRNA-3 plasmid showed a satisfactory interfering effect.After TPX2 gene was silenced effectively,both the transcription of TPX2 mRNA and expression of TPX2 protein in A549 cells were inhibited significantly（P 〈 0.01）,with inhibitory rates of 73.8% and 82.8% as compared with those in control group respectively.Both the transcription levels CDK4 and CyclingD1 mRNAs decreased significantly（P 〈 0.01 or P 〈 0.05）,with inhibitory rates of 76.7% and 67.3% as compared with those in control group respectively.The growth and proliferation of A549 cells were inhibited significantly,of which the inhibitory rate 72 h after transfection was 61.55%（P 〈 0.05）.Conclusion The shRNA plasmid for TPX2 gene was successfully constructed,which inhibited the expression of TPX2 at both mRNA and protein levels in A549 cells,afterwards the growth and proliferation of A549 cells were inhibited.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第7期848-851,共4页
Chinese Journal of Biologicals
