【摘要】 目的:构建Snail的慢病毒真核表达载体,并研究Snail在肿瘤发生和发展过程中对上皮-间质转化(EMT)的影响。方法:以乳腺文库为模板,PCR扩增Snail的全长编码序列,将其克隆到p CDH表达载体上,构建成p CDH-Snail真核表达载体,转染293T细胞,Western印迹检测p CDH载体介导的Snail的表达;与包装质粒共转染293T细胞,包装病毒后感染ZR75-1细胞,经嘌呤霉素筛选2周,得到稳定表达Snail的ZR75-1细胞株,检测ZR75-1细胞对EMT的影响,同时用倒置荧光显微镜观察ZR75-1稳定细胞株中上皮钙粘蛋白分布的变化。结果:Bam HⅠ、XbaⅠ双酶切及测序证实得到p CDH-Snail表达载体,Western印迹表明Snail在293T细胞内得到成功;经嘌呤霉素筛选,获得稳定表达Snail的ZR75-1细胞株;在倒置荧光显微镜下观察,ZR75-1稳定细胞株中的上皮钙粘蛋白绿色荧光明显减弱。结论:构建了p CDH-Snail的慢病毒真核表达载体,建立了稳定表达Snail的ZR75-1细胞,为进一步研究Snail在EMT过程中的机制奠定了基础。更多还原

【Abstract】 Objective: To construct an eukaryotic expression vector of human Snail gene and to detect effect onepithelial-mesenchymal transition(EMT). Methods: The coding sequences of full length Snail was amplified frombreast library by PCR and cloned into the p CDH vector. Snail expression was detected by Western blot after therecombinant plasmids were transfected into 293 T cells. p CDH-Snail was then packaged with accessory plasmids in-to lentivirus in 293 T cells and selected for 2 weeks with puromycin before the mixed colonies stably expressingSnail were obtained. The fluorescent signals were detected by fluorescence microscope. Results: The full lengthSnail was expressed in the ZR75-1 cells. Compared to the empty vector, the expression of E-cadherin was de-creased by Snail. Conclusion: The eukaryotic expression vector of Snail was successfully constructed and ex-pressed in human ZR75-1 cells, and lay foundation for further research on Snail mechanism in breast cancer cellEMT.更多还原