siRNA抑制cortactin基因表达对喉癌Hep-2细胞增殖和侵袭的影响

发布时间：2018-06-30 03:51

本文选题：cortactin + 喉癌　；参考：《南华大学》2010年硕士论文

【摘要】： 背景与目的:皮层肌动蛋白结合蛋白cortactin (cortical actin-binding protein)参与细胞骨架系统的调控、细胞外信号转导以及细胞黏附等过程,研究表明cortactin与肿瘤的侵袭和转移有关。然而,cortactin在喉癌中的重要作用还没有被详细的阐明。本实验探讨siRNA(small interfering RNA)抑制cortactin基因表达对喉癌Hep-2细胞系体外增殖和侵袭的影响。方法:利用基因重组技术构建针对cortactin基因的小干扰RNA真核表达载体pSilencer3.1-H1neo-cortactin;用DNA测序法鉴定重组质粒;用脂质体转染技术将重组质粒转染Hep-2细胞,同时以转空载体组和空白组作为对照(三组细胞分别命名为pcortactin-siRNA/Hep-2,pSilencer3.1/Hep-2,Hep-2),G418筛选出阳性细胞克隆;应用Western blot及免疫细胞化学方法分别检测cortactin在Hep-2中的siRNA干扰效率及定位;MTT和平皿克隆形成实验检测细胞增殖能力;流式细胞仪检测细胞周期分布情况;Transwell体外迁移、侵袭实验检测细胞迁移侵袭能力。结果: DNA测序分析表明针对cortactin基因的重组真核表达载体pSilencer3.1-H1neo-cortactin构建成功。免疫细胞化学方法显示cortactin在Hep-2细胞胞浆中呈弥漫性表达,Western blot显示成功构建cortactin表达下调的稳转喉癌细胞系pcortactin- siRNA/Hep-2。与空白组Hep-2细胞比较, pcortactin-siRNA/Hep-2的cortactin含量为11.22%(P0.01);细胞生长速度明显减慢(P0.05);克隆形成率降低为21.47% (P0.01);细胞中S期细胞百分比明显降低(P0.05);迁移、侵袭能力显著降低(P0.01)。结论:1.成功建立cortactin表达下调的稳定转染喉癌细胞系pcortactin-siRNA/Hep-2。 2.Cortactin表达下调能抑制喉癌Hep-2细胞的增殖和侵袭。
[Abstract]:Background & AIM: cortical actin binding protein (cortactin (cortical actin-binding protein) is involved in the regulation of cytoskeleton system, extracellular signal transduction and cell adhesion. However, the important role of cortactin in laryngeal carcinoma has not been elucidated in detail. The aim of this study was to investigate the effects of siRNA (small interfering on the proliferation and invasion of laryngeal cancer cell line Hep-2 in vitro. Methods: the small interfering cortactin eukaryotic expression vector pSilencer3.1-H1 neo-cortacin was constructed by gene recombination technique, the recombinant plasmid was identified by DNA sequencing, and the recombinant plasmid was transfected into Hep-2 cells by liposome transfection technique. At the same time, the positive cell clones were screened by G418 in the empty vector group and blank group (the three groups were named pcortactin-siRNA-pcortactin-siRNA / Hep-2pSilencer3.1 / Hep-2 / Hep-2) respectively. Western blot and immunocytochemistry were used to detect the siRNA interference efficiency of cortactin in Hep-2 and the ability of cell proliferation to detect the cell proliferation, and the flow cytometry was used to detect the cell cycle distribution and the migration of cortactin in vitro. Invasion assay was used to detect the ability of cell migration and invasion. Results: DNA sequencing analysis showed that the recombinant eukaryotic expression vector pSilencer3.1-H1neo-cortactin was successfully constructed. Immunocytochemistry showed that cortactin was diffusely expressed in the cytoplasm of Hep-2 cells. Western blot showed that pcortactinsiRNA-siRNA / Hep-2 cell line with down-regulated cortactin expression was successfully constructed. Compared with the control group, the cortactin content of pcortactin-siRNA / Hep-2 was 11.22% (P0.01), the cell growth rate was significantly slower (P0.05), the clone formation rate was reduced to 21.47% (P0.01), the percentage of S phase cells was significantly decreased (P0.05), the migration and invasion ability were significantly decreased (P0.01). Conclusion 1. A stable transfected laryngeal carcinoma cell line pcortactin-siRNA / Hep-2.2.Cortactin down-regulated can inhibit the proliferation and invasion of laryngeal carcinoma cell line Hep-2.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65

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