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

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

【參考文獻】

相關期刊論文 前6條

1 黃志剛;;喉癌基礎研究現(xiàn)狀和展望[J];中國耳鼻咽喉頭頸外科;2010年02期

2 李連賀;岳卓立;馮秀玲;劉吉娜;劉淑紅;;EMS1蛋白在喉癌組織中的表達及臨床意義[J];臨床耳鼻咽喉頭頸外科雜志;2009年15期

3 高國政;趙瑞力;張笑穎;郭艷麗;楊植彬;董稚明;;喉鱗狀細胞癌中FAK和E-cadherin的表達及意義[J];臨床與實驗病理學雜志;2009年04期

4 李跟紅;朱春生;;RNA干擾及其在喉癌基因治療中的應用現(xiàn)狀[J];實用醫(yī)藥雜志;2009年07期

5 林敏;謝海龍;蘇琦;;EMS1基因與腫瘤[J];國際病理科學與臨床雜志;2005年06期

6 ;CTTN (EMS1): an oncogene contributing to the metastasis of esophageal squamous cell carcinoma[J];Cell Research;2007年04期

相關博士學位論文 前1條

1 郜永光;亨廷頓蛋白中脯氨酸豐富區(qū)域與SH3和雙WW結構域的相互作用[D];中國科學院研究生院（上海生命科學研究院）;2006年

，

本文編號：2084747