转染Lgr5基因对结肠癌细胞株体外生物学特性影响的研究  

The preliminary functional study of a Lgr5-harboring SW480 cell line

作　　者：高菲[1] 傅丰庆[2] 徐俊驰[3] 高兵[1] 胡玉敏[1] 张学光[2] 

机构地区：[1]江苏省干细胞重点实验室、苏州大学医学生物技术所,215007 [2]苏州大学附属第一医院江苏省临床免疫研究所 [3]苏州第五人民医院

出　　处：《国际免疫学杂志》2013年第3期216-220,共5页International Journal of Immunology

基　　金：国家自然科学基金（30901313）

摘　　要：目的构建pIRES2-EGFP-Lgr5重组表达载体，获得SW480／Lgr5瞬时基因转染细胞株，并探讨转染Lgr5基因对结肠癌细胞株生物学特性的影响。方法运用逆转录聚合酶链式反应（RT—PCR）技术从结肠癌组织中获得Lgr5全长基因，经双酶切（XhoI和BamHI）连接入真核表达载体pIRES2-EGFP中，利用脂质体法转染SW480细胞，经流式细胞术、Westernblot、免疫细胞化学法检测Lgr5分子的表达；CCK-8法和悬滴实验分析Lgr5对结肠癌细胞增殖率和成球能力的影响，划痕实验分析Lgr5对结肠癌细胞迁移能力的影响。结果成功构建真核表达载体pIRES2。EGFP／hLgr5，并获得SW480／Lgr5瞬时基因转染细胞株；转染Lgr5基因的结肠癌细胞体外增殖速度加快，形成的细胞球大而紧密但迁移能力下降。结论Lgr5基因转染促进结肠癌细胞株体外的生长，为进一步研究人Lgr5分子的生物学特性及其在结肠癌中的作用机制奠定了基础。Objective To consmact the pIRES2-EGFP-Lgr5 recombinant expression vector, establish Lgr5 transient transfected SW480 cells, and investigate the effect of Lgr5 overexpression on the biological char- acteristic of colon cancer cells. Methods cDNA fragment encoding human Lgr5 gene was amplified from hu- man colon cancer tissue by reverse transcription-PCR. After Lgr5 gene was dissected with the restriction endo- nucleases XhoI and BamHI, it was inserted into pIRES2-EGFP vector. The recombinant pIRES2-EGFP/Lgr5 was then tranfected into SW480 cells by fipofectamine 2000. The expression of Lgr5 in the transfected cells was identified by flow cytometric analysis, Western blot and immunohistochemistry. The proliferation and the ability o~ packing into spheroids of the transfected cells in present and in the absence of cell matrix were detected by CCK-8 assay and hanging drops assay, while migration of the cells was analyzed by the scratch test. Results The recombinant plRES2-EGFP/Lgr5 expression vector was constructed and transient transfected SW480 cell line with Lgr5 overexpression was established. Colon cancer cells with Lgr5 overexpression grew faster, packed into more compact spheroids resistant to mechanical disruption and had a weaker ability to migrate compared to the cells infected control vector. Conclusion The research has provided solid experiment foundation for further studies in the biological characteristic of Lgr5 and its role in the development of colon cancer.

关 键 词：Lgr5基因 真核表达载体 转染 SW480细胞 生物学特性 

分 类 号：R735.35[医药卫生—肿瘤]