引用本文:胡 萍,杨佳佳,黄 翼,曾宗跃,唐 曦,黄杰涛,柳满然.转录因子Twist基因慢病毒质粒构建及其促进乳腺肿瘤上皮细胞MCF-7上皮间质转化[J].重庆医科大学学报,2014,38(3):328~331
转录因子Twist基因慢病毒质粒构建及其促进乳腺肿瘤上皮细胞MCF-7上皮间质转化
Construction of lentivirus expression vector containing transcription factor Twist gene and its effect on promoting epithelial to mesenchymal transition of breast cancer cells MCF-7
DOI:
中文关键词:  Twist  慢病毒  MCF-7  侵袭  上皮间质转化
英文关键词:Twist  lentivirus  MCF-7  invasion  epithelial to mesenchymal transition
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胡 萍,杨佳佳,黄 翼,曾宗跃,唐 曦,黄杰涛,柳满然 重庆医科大学临床检验诊断学教育部重点实验室重庆 400016 
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中文摘要:
      目的:构建转录因子Twist基因的慢病毒质粒,其高表达促进乳腺肿瘤上皮细胞MCF-7上皮间质转化(epithelial to mesenchymal transition,EMT)。方法:pcDNA3/myc-tagged-Twist质粒扩增并亚克隆至载体pLVX-puro中构建成pLVX-puro-myc-tagged-Twist慢病毒质粒。将其转染人胚肾细胞HEK293T,收取病毒上清感染乳腺癌MCF-7细胞。免疫荧光法和蛋白质印迹法(Western blot)检测转录因子Twist 蛋白、上皮标志蛋白E-钙黏蛋白(E-cadherin)、间质标志蛋白波形蛋白(Vimentin)的表达。Transwell法检测细胞侵袭能力。结果:pLVX-puro-myc-tagged-Twist慢病毒质粒酶切及测序结果完全正确;相对于对照细胞(MCF-7-Vector细胞),转染Twist基因的MCF-7细胞(MCF-7-Twist细胞)Twist蛋白、间质标志蛋白Vimentin表达水平明显升高,上皮标志蛋白E-cadherin表达水平明显降低(P<0.05),侵袭能力显著增强(P<0.05)。结论:pLVX-puro-myc-tagged-Twist慢病毒质粒构建成功,转录因子Twist通过MCF-7细胞上皮间质转化促进了乳腺肿瘤侵袭能力增强。
英文摘要:
      Objective:To construct lentivirus expression vector containing Twist gene and to explore its overexpression induced epithe-lial to mesenchymal transition(EMT) in MCF-7 of breast cancer cells. Methods:pcDNA3/myc-tagged-Twist plasmid was amplified by PCR and then cloned into pLVX-puro-vector. After the construction being transfected into HEK-293 cells,the supernatant was collected to infect the MCF-7 cells. The expression of Twist E-cadherin and Vimentin was analyzed by immunofluorescence and Western blot. The invasive ability was evaluated by Transwell invasion assay in MCF-7-Twist cells. Results:The pLVX-puro-myc-tagged-Twist was confirmed correctly by DNA sequencing. Compared with that of vector control cells,the expression of Twist,E-cad-herin and Vimentin was much higher and the invasive ability was much stronger in MCF-7-Twist cells(P<0.05). Conclusions:The recombinant lentivirus vector expressing Twist is constructed and is efficaciously expressed in MCF-7 cells,which could significantly promote the invasion ability of MCF-7 cells through EMT.
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