摘要
目的构建Twist基因逆转录病毒载体,研究其对人正常乳腺上皮细胞MCF10A的影响。方法酶切pcDNA3/mycTwist获得myc-Twist,克隆到逆转录病毒载体pBABE-puro中,构建重组质粒pBABE-myc-Twist。通过酶切、测序鉴定Twist基因正确后,在体外将质粒pBABE-myc-Twist和其对照分别与包装质粒pAmpho共同转染人胚肾上皮细胞系293T细胞,进行逆转录病毒的包装,并感染人正常乳腺MCF10A上皮细胞,用嘌呤霉素(puromycin)筛选,获得成功转入Twist的MCF10A-Twist细胞和MCF10A-Vector对照细胞。通过RT-PCR和Western blot法验证Twist细胞在MCF10A-Twist和MCF10A-Vector细胞内的表达。免疫荧光技术和Western blot法检测细胞内上皮间质转化(EMT)标志蛋白的表达。Transwell^(?)法检测细胞的迁移和侵袭能力。结果重组逆转录病毒载体质粒pBABE-myc-Twist经酶切和测序鉴定构建正确;Twist基因被逆转录病毒成功导入MCF10A细胞,并在靶细胞内稳定表达,RT-PCR检测到Twist mRNA在MCF10A-Twist细胞中表达,Western blot法检测发现myc-Twist蛋白在MCF10A-Twist细胞中表达;免疫荧光和Western blot法检测显示在MCF10A-Twist细胞中E-cadherin表达显著下调、vimentin表达明显上调;与MCF10A-Vector细胞相比,Transwell^(?)法检测发现MCF10A-Twist细胞迁移和侵袭能力明显增加(P<0.01)。结论 Twist诱导MCF10A细胞发生EMT转化,并促进其迁移和侵袭。
Objective To construct a retroviral vector carrying Twist gene and investigate its effect on human mammary MCFIOA epithelial cells. Methods Myc-Twistwas digested from pcDNA3/myc-Twist and subcloned into the retroviral vector pBABE-puro to construct a recombinant plasmid （pBABE-myc-Twist）. The inserted Twist gene was confirmed by restriction enzyme digestion and DNA sequencing. The plasmid pBABE-myc-Twist and the packaging plasmid pAmpho were co-trans- fected into HEK？.93T cells for packaging of retrovirus. Meanwhile, the control plasmid pBABE-puro and the packaging plasmid were co-transfected into the other HEK293T cells as a control group. Human mammary MCF10A epithelial cells were infected with the retroviruses carrying Twist gene or the controls, and selected by puromycin. The expression of Twist in the MCF10A- Twist and MCF10A-Vector cells was determined by RT-PCR and Western blotting. The expressions of epithelial-mesenchymal tran- sition （EMT） marker proteins induced by Twist in MCFIOA cells were detected using immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were analyzed by Transwell~ assay. Results The myc-tagged Twist gene was correctly inserted into the retroviral expression vector as a recombinant plasmid （pBABE-myc-Twist） as identified by restriction analysis and DNA sequencing. The Twist gene was efficiently delivered into human mammary MCF10A epithelial cells by the retrovirus, resulting in the stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT - PCR and Western blotting, respectively. The expression ofthe epithelial biomarker E - cadherin was downregulated whereas, the masenchyrnal marker vimentin upregulated in MCF10A-Twist cells as shown by immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were enhanced notably in MCF10A-Twist cells as compared with MCF10A-Vector control cells （ P 〈 0.01 ）. Conclusion Twist induces EMT of MCF10A cells and plays an important role in
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第9期905-909,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31171336)
高校博士点基金(20105503110001
20125503110001)
教育部留学回国启动基金(教外司[2011]508号)
