發(fā)布時(shí)間：2018-06-30 03:51

  本文選題：cortactin + 喉癌��； 參考：《南華大學(xué)》2010年碩士論文

【摘要】： 背景與目的:皮層肌動(dòng)蛋白結(jié)合蛋白cortactin (cortical actin-binding protein)參與細(xì)胞骨架系統(tǒng)的調(diào)控、細(xì)胞外信號轉(zhuǎn)導(dǎo)以及細(xì)胞黏附等過程,研究表明cortactin與腫瘤的侵襲和轉(zhuǎn)移有關(guān)。然而,cortactin在喉癌中的重要作用還沒有被詳細(xì)的闡明。本實(shí)驗(yàn)探討siRNA(small interfering RNA)抑制cortactin基因表達(dá)對喉癌Hep-2細(xì)胞系體外增殖和侵襲的影響。 方法:利用基因重組技術(shù)構(gòu)建針對cortactin基因的小干擾RNA真核表達(dá)載體pSilencer3.1-H1neo-cortactin;用DNA測序法鑒定重組質(zhì)粒;用脂質(zhì)體轉(zhuǎn)染技術(shù)將重組質(zhì)粒轉(zhuǎn)染Hep-2細(xì)胞,同時(shí)以轉(zhuǎn)空載體組和空白組作為對照(三組細(xì)胞分別命名為pcortactin-siRNA/Hep-2,pSilencer3.1/Hep-2,Hep-2),G418篩選出陽性細(xì)胞克隆;應(yīng)用Western blot及免疫細(xì)胞化學(xué)方法分別檢測cortactin在Hep-2中的siRNA干擾效率及定位;MTT和平皿克隆形成實(shí)驗(yàn)檢測細(xì)胞增殖能力;流式細(xì)胞儀檢測細(xì)胞周期分布情況;Transwell體外遷移、侵襲實(shí)驗(yàn)檢測細(xì)胞遷移侵襲能力。 結(jié)果: DNA測序分析表明針對cortactin基因的重組真核表達(dá)載體pSilencer3.1-H1neo-cortactin構(gòu)建成功。免疫細(xì)胞化學(xué)方法顯示cortactin在Hep-2細(xì)胞胞漿中呈彌漫性表達(dá),Western blot顯示成功構(gòu)建cortactin表達(dá)下調(diào)的穩(wěn)轉(zhuǎn)喉癌細(xì)胞系pcortactin- siRNA/Hep-2。與空白組Hep-2細(xì)胞比較, pcortactin-siRNA/Hep-2的cortactin含量為11.22%(P0.01);細(xì)胞生長速度明顯減慢(P0.05);克隆形成率降低為21.47% (P0.01);細(xì)胞中S期細(xì)胞百分比明顯降低(P0.05);遷移、侵襲能力顯著降低(P0.01)。 結(jié)論:1.成功建立cortactin表達(dá)下調(diào)的穩(wěn)定轉(zhuǎn)染喉癌細(xì)胞系pcortactin-siRNA/Hep-2。 2.Cortactin表達(dá)下調(diào)能抑制喉癌Hep-2細(xì)胞的增殖和侵襲。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R739.65

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