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目的在肿瘤发生和发展过程中,上皮细胞-间质转化(epithelial-mesenchymal transition,EMT)是上皮来源恶性肿瘤细胞获得侵袭、转移能力的重要生物学过程,并且与乳腺癌干细胞的生物学特性密切相关。本研究通过构建Slug的慢病毒真核表达载体,探讨Slug基因与EMT之间的关系及对乳腺癌干细胞生物学特性的影响。方法以军事医学科学院(生物工程研究所)保存的乳腺cDNA文库为模板,通过PCR扩增出Slug的全长编码序列,将其克隆到经BamHⅠ和EcoRⅠ酶切的PCDH表达载体上,构建成PCDH-Slug真核表达载体,在人293T细胞中转入空载体和重组质粒,利用蛋白质印迹法检测PCDH载体介导的Slug的表达。后将包装质粒共转染293T细胞,包装病毒,感染ZR75-1细胞,经Puro(嘌呤霉素)筛选2周后,得到稳定表达Slug的ZR75-1细胞株,检测ZR75-1细胞对EMT的影响。并将稳定表达Slug的ZR75-1稳定细胞接种于的Matrigel基质胶,倒置显微镜观察ZR75-1稳定细胞株的成管特性。结果从乳腺文库中获得Slug的全长编码序列,经BamHⅠ、EcoRⅠ双酶切及DNA测序证实得到PCDH-Slug表达载体,PCDH-Slug真核表达载体和PCDH分别转染293T细胞后,经过蛋白质印迹法检测Slug在293T细胞内有显影,对照组明显减弱,表明Slug-PCDH载体携带的Slug编码序列能够在293T细胞中成功;通过增加puro的含量进行筛选,得到稳定表达的混合克隆即能表达Slug的ZR75-1细胞株,经蛋白质印迹法检测PCDH-Slug对E-cadherin的影响,以GAPDH为内参,结果比对照组的表达减弱,与预期相符,表明PCDH-Slug载体可以使细胞中E-cadherin的表达减弱;倒置显微镜下ZR75-1稳定细胞株成管明显,表明Slug可以通过调节EMT使乳腺癌细胞具有某些干性特征。结论调节Slug基因表达可以影响EMT相关蛋白的表达,并以此影响乳腺癌干细胞的生物学特性。
OBJECTIVE: Epithelial-mesenchymal transition (EMT) is an important biological process of invasion and metastasis of epithelial-derived malignant tumor cells in tumorigenesis and progression, and is closely related to the biological characteristics of breast cancer stem cells Related. In this study, Slug lentiviral eukaryotic expression vector was constructed to investigate the relationship between Slug gene and EMT and the biological characteristics of breast cancer stem cells. Methods The full-length coding sequence of Slug was amplified by PCR from the breast cDNA library preserved by the Academy of Military Medical Sciences (Institute of Bioengineering) and cloned into the PCDH expression vector digested with BamHⅠ and EcoRⅠ to construct PCDH -Slug eukaryotic expression vector was transfected into empty vector and recombinant plasmid in human 293T cells. Western blotting was used to detect PCDH vector-mediated Slug expression. The packaged plasmid was cotransfected into 293T cells, packaged and infected with ZR75-1 cells. After puro (puromycin) screening for 2 weeks, ZR75-1 cell line stably expressing Slug was obtained, and the effect of ZR75-1 cells on EMT influences. The ZR75-1 stable cells stably expressing Slug were inoculated on Matrigel matrices, and the tube formation characteristics of the ZR75-1 stable cell lines were observed under an inverted microscope. Results The full-length coding sequence of Slug was obtained from the mammary gland library. The PCDH-Slug expression vector was confirmed by double enzyme digestion with BamHⅠ and EcoRⅠ and DNA sequencing. PCDH-Slug eukaryotic expression vector and PCDH were transfected into 293T cells respectively, Slug was detected in 293T cells and the control group was significantly weakened, indicating that the Slug coding sequence carried by Slug-PCDH vector could be successfully transfected into 293T cells. By screening purpurin, the stable expression clones expressing Slug Of ZR75-1 cells, Western blotting detection of PCDH-Slug on E-cadherin, GAPDH as an internal control, the results of the expression of the control group weakened, in line with expectations, indicating that the PCDH-Slug vector cells can make E- cadherin expression weakened; inverted microscope ZR75-1 stable cell line tube obvious, indicating that Slug EMF can make breast cancer cells have some dry features. Conclusion Regulation of Slug gene expression can affect the expression of EMT-related proteins and thus affect the biological characteristics of breast cancer stem cells.