稳定表达Notch1胞内结构域肺腺癌细胞株的构建及鉴定  

Construction and identification of lung adenocarcinoma cell line with stable expression of N1ICD

作　　者：邹斌[1] 吴霞[1] 周学亮[1] 詹宇亮 赖松青[1] 刘季春[1] 

机构地区：[1]南昌大学第一附属医院,南昌330006

出　　处：《山东医药》2017年第6期5-8,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81570262);江西省研究生创新专项资金(YC2015-B009)

摘　　要：目的构建Notch1胞内结构域(N1ICD)稳定表达的肺腺癌A549细胞株。方法采用PCR方法扩增N1ICD基因,构建慢病毒重组质粒p HBLV-N1ICD-Zs Green-Puro(LV-N1ICD),采用PCR和基因测序鉴定重组质粒。高纯度无内毒素抽提慢病毒重组质粒和辅助包装原件载体质粒,使用慢病毒空载质粒(LV-Zs Green-Puro)和LVN1ICD共转染293T细胞包装慢病毒,利用药物筛选法测定病毒滴度。将A549细胞分成A549-N1ICD组和A549-Zs Green-Puro组,分别加入LV-N1ICD和LV-Zs Green-Puro,取制备好的慢病毒感染A549细胞,加入嘌呤霉素筛选稳定转染细胞株,荧光显微镜观察转染效率,以带有绿色荧光的细胞初步视为稳定转染的细胞株。以A549细胞为对照组,进一步采用q PCR和Western blot方法分别检测N1ICD mRNA及蛋白表达。结果 PCR及基因测序结果表明pHBLV-N1ICD-Zs Green-Puro重组质粒构建成功。LV-N1ICD组病毒滴度为1×10~8TU/m L,LV-Zs Green-Puro组病毒滴度为1×108TU/m L。经嘌呤霉素筛选后,荧光显微镜下观察细胞形态无明显变化,A549-Zs Green-Puro组和A549-N1ICD组70%~80%的细胞带有绿色荧光。对照组、A549-Zs Green-Puro组、A549-N1ICD组的N1ICD mRNA相对表达量分别为1.59±0.11、1.09±0.10、70.81±6.39,A549-N1ICD组高于其他两组(P均<0.01)。三组N1ICD蛋白相对表达量分别为1.99±0.13、1.73±0.08、6.58±0.43,A549-N1ICD组高于其他两组(P均<0.01)。结论成功构建了稳定表达N1ICD的肺腺癌A549细胞株。Objective To construct a lung adenocarcinoma cell line A549 with stable expression of Notch1 intracellular domain（N1ICD）.Methods The N1 ICD fragment was amplified by PCR,which was used to construct p HBLVN1ICD-Zs Green-Puro plasmid（LV-N1ICD）.The recombinant plasmid was identified by PCR and gene sequencing.LVN1 ICD was packaged by transfection of 293 T cells with recombinant plasmid and auxiliary plasmids which were purified without endotoxin extraction.Lentivirus empty plasmid（LV-Zs Green-Puro） was also constructed.Drug screening method was used to determine the titer of LV-N1 ICD and LV-Zs Green-Puro.A549 cells were divided into A549-N1 ICD group and A549-Zs Green-Puro group.Puromycin was used to screen stable transfection cell line after infection of A549 cell with LVN1 ICD or LV-Zs Green-Puro.Transfection efficiency was observed by fluorescence microscope and cells with green fluorescent were preliminarily identified as stable transfection cell lines.Taking A549 cells as the control group,and N1ICD mRNA and protein expression was further detected by q PCR and Western blotting,respectively.Results PCR and gene sequencing showed that p HBLV-N1ICD-Zs Green-Puro plasmid was successfully constructed.The titer of LV-N1 ICD was 1×10^8TU/m L and the LV-Zs Green-Puro was 1× 10^8 TU/m L.Cells shape had no obvious changes and green fluorescence was detected in about 70%-80% cells of the A549-Zs Green-Puro and A549-N1 ICD group by fluorescence microscope after Puromycin screening.The N1ICD mRNA expression of the control group,A549-Zs Green-Puro group and A549-N1 ICD group was 1.59±0.11,1.09±0.10 and 70.81±6.39,respectively and the N1ICD mRNA expression of the A549-N1 ICD group was significant higher than that of the other two groups（all P〈0.01）.The N1 ICD protein expression of the three groups was 1.99±0.13,1.73±0.08 and 6.58±0.43,respectively and the N1ICD protein expression of the A549-N1 ICD group was significant higher than that of the other two groups（all P〈0.01）.Conclusions A lung ad

关 键 词：肺腺癌 Notch蛋白 胞内结构域 慢病毒重组质粒 

分 类 号：R734.2[医药卫生—肿瘤]