目的 研究干扰素调节因子4结合蛋白(IBP)的表达抑制对人乳腺癌MDA-MB-231细胞中微丝、微管的影响.方法 设计并合成针对人IBP mRNA的RNA干扰(RNAi)序列,构建RNAi慢病毒表达载体,与包装质粒共转染人胚肾293FT细胞获得病毒颗粒,以病毒感染MDA-MB-231细胞.采用免疫印迹及实时聚合酶链反应(RT-PCR)确定能有效抑制IBP的RNAi序列,筛选出获得稳定感染的细胞株并进行微丝、微管染色.结果 成功构建了IBP特异性的RNAi慢病毒表达载体和对照载体,对MDA-MB-231细胞IBP的沉默效率为69.9%;通过微丝、微管染色证实IBP表达下调后,细胞形态出现明显变化,细胞体增大,丝状伪足减少,片状伪足增多,微管排列紊乱.结论 IBP蛋白通过影响微管、微丝的排列而改变MDA-MB-231细胞生物学行为.%Objective To study the effects of interferon regulatory factor-4 binding protein(IBP)expression inhibition on mi-crofilament and microtubule of human breast cancer MDA-MB-231 cells .Methods RNA interference (RNAi) sequences targeting human IBP mRNA were designed and synthesized .Lentiviral vectors expression RNAi were constructed and then were co-trans-fected with packaging plasmids into human embryonic kidney 293FT cells to obtain virus particles for infection of MDA-MB-231 cells .Western blot and real time-polymerase chain reaction(RT-PCR) were employed to confirm the sequences which could inhibit IBP effectively.Cells with stable infection were screened out and then their microfilament and microtubule were subjected to stai -ning.Results Lentiviral vector expression IBP-specific RNAi and control vector were constructed successfully .IBP-silence efficiency in MDA-MB-231 cells was 69 .9%.Microfilament and microtubule staining confirmed that IBP expression in cells were down-regulated.Obvious morphological changes of cells were observed ,such as increasing cell body ,decreasing filopodia ,increasing lamel-lipodia and disorder of microtubule arrangement.Conclusion IBP protein alter the biological behavior of MDA-MB-231 cells via influencing the arrangement of microtubules and microfilaments .
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