人AURKA基因RNA干扰慢病毒载体的构建及其对A549细胞增殖的影响

发布时间：2018-06-22 11:39

本文选题：病毒 + AURKA基因　；参考：《蚌埠医学院学报》2016年11期

【摘要】：目的:针对AURKA基因构建RNA干扰重组慢病毒质粒并进行慢病毒包装,初步探讨AURKA基因的功能。方法:应用p GCLV-GFP慢病毒载体构建针对AURKA的shRNA载体,转染包装293T细胞,收集病毒上清液,转染肺腺癌细胞株A549。荧光显微镜下观察绿色荧光蛋白表达情况确定转染效率,应用Western blot技术检测AURKA基因在蛋白水平表达的变化,以明确RNAi的抑制率。应用Brd U法分析干扰AURKA前后A549细胞增殖情况的变化,运用克隆形成实验检测干扰AURKA后A549细胞株对长春新碱敏感性的变化。结果:针对AURKA基因的RNAi慢病毒表达载体构建成功,PCR和DNA测序鉴定实验表明插入位点与干扰片断的碱基序列完全正确。在293T细胞中进行慢病毒包装,获得高滴度病毒上清液。通过重组慢病毒载体将AURKA shRNA转导入A549细胞中,转染效率为100%。Western blot检测证实LV-AURKA慢病毒显著抑制AURKA的表达,Brd U检测显示AURKA基因表达下调抑制了A549细胞的增殖。克隆形成实验表明AURKA基因表达增强A549细胞对长春新碱的敏感性。结论:慢病毒载体介导的靶向AURKA的RNA干扰可有效抑制AURKA表达,降低肺腺癌A549细胞的增殖能力,并且增强A549细胞对长春新碱的敏感性。
[Abstract]:Aim: to construct recombinant lentivirus plasmid with RNA interference targeting AURKA gene and to investigate the function of AURKA gene. Methods: pGCLV-GFP lentivirus vector was used to construct shRNA vector for AURKA. The recombinant plasmid was transfected into 293T cells. The supernatant was collected and transfected into lung adenocarcinoma cell line A549. The transfection efficiency was determined by observing the expression of green fluorescent protein under fluorescence microscope, and the changes of Aurka gene expression at protein level were detected by Western blot to determine the inhibition rate of RNAi. Brd-U assay was used to analyze the proliferation of A549 cells before and after AURKA interference, and the sensitivity of A549 cells to vincristine was detected by clone formation assay. Results: the RNAi lentivirus expression vector of Aurka gene was successfully constructed and identified by PCR and DNA sequencing. The supernatant of high titer virus was obtained by packaging lentivirus in 293T cells. AURKA shRNA was transfected into A549 cells by recombinant lentivirus vector. The transfection efficiency was 100%. Western blot assay confirmed that LV-AURKA lentivirus significantly inhibited the expression of AURKA. Brd U assay showed that AURKA gene expression down-regulated the proliferation of A549 cells. Cloning assay showed that AURKA gene expression enhanced the sensitivity of A549 cells to vincristine. Conclusion: lentivirus vector mediated RNA interference targeting AURKA can effectively inhibit the expression of AURKA, reduce the proliferation of lung adenocarcinoma A549 cells, and enhance the sensitivity of A549 cells to vincristine.
【作者单位】：南通大学附属吴江医院胸外科;江苏大学附属昆山医院胸外科;南通大学第四附属医院胸外科;苏州大学医学部;
【基金】：苏州市科学技术计划项目(SYS201301) 昆山市科学基金项目(ks1234)
【分类号】：R734.2;Q78

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