發(fā)布時(shí)間：2018-03-07 00:06

  本文選題：Aurora激酶　切入點(diǎn)：喉腫瘤　出處：《復(fù)旦大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：目的：研究Aurora激酶A (Aurora Kinase A, AURKA)在喉鱗狀細(xì)胞癌中的表達(dá)及其臨床意義,研究AURKA基因沉默在體外體內(nèi)對喉癌HEp-2細(xì)胞生長的影響及其機(jī)制。 方法：收集37例喉鱗狀細(xì)胞癌組織和鄰近正常組織,應(yīng)用實(shí)時(shí)熒光RT-PCR和Western blot技術(shù)檢測AURKA mRNA和蛋白在喉癌及鄰近正常組織中的表達(dá),分析AURKA mRNA和蛋白與喉癌臨床病理特征的關(guān)系。 構(gòu)建針對AURKA的shRNA表達(dá)質(zhì)粒并轉(zhuǎn)染喉鱗癌細(xì)胞株HEp-2細(xì)胞,檢測HEp-2細(xì)胞轉(zhuǎn)染前后AURKA mRNA和蛋白表達(dá),CCK-8實(shí)驗(yàn)檢測細(xì)胞增殖,Transwell實(shí)驗(yàn)檢測細(xì)胞侵襲,軟瓊脂實(shí)驗(yàn)觀察克隆形成能力,流式細(xì)胞術(shù)檢測細(xì)胞周期分布、凋亡和有絲分裂監(jiān)控點(diǎn),免疫熒光實(shí)驗(yàn)觀察細(xì)胞間橋、多極紡錘體和多中心體等染色體不穩(wěn)定性事件,Western blot檢測細(xì)胞粘附和遷移的重要因子粘著斑激酶(FAK)和基質(zhì)金屬蛋白酶-2(MMP-2)蛋白表達(dá)。另外,將AURKA基因敲除的HEp-2細(xì)胞(HEp-2-S)接種于裸鼠,觀察裸鼠移植瘤生長情況以及瘤體內(nèi)FAK和MMP-2的表達(dá),最后,將AURKA抑制劑VX-680注射入荷瘤裸鼠,觀察VX-680對腫瘤生長的影響。 結(jié)果：32例(86.5%)病人腫瘤中的AURKA mRNA高于相應(yīng)的喉正常黏膜,AURKA mRNA在喉癌組織中表達(dá)上調(diào),顯著高于喉正常黏膜組織中的表達(dá)。25對喉癌組織及其喉正常黏膜中,其中有16個(gè)(64.0%)病例AURKA蛋白T/N比值1.2,提示在喉癌組織中AURKA蛋白呈高表達(dá)。AURKA mRNA上調(diào)和蛋白的高表達(dá)與患者頸部淋巴結(jié)轉(zhuǎn)移顯著相關(guān),AURKA mRNA還與臨床Ⅲ/Ⅳ分期顯著相關(guān),而與患者性別、年齡、腫瘤部位、T分期和病理分級無相關(guān)性。喉癌HEp-2細(xì)胞中AURKA蛋白也呈高表達(dá)。 HEp-2細(xì)胞轉(zhuǎn)染AURKA shRNA后,AURKA mRNA和蛋白表達(dá)明顯下降。AURKA沉默后,抑制HEp-2細(xì)胞增殖活性、侵襲和克隆形成能力,以及裸鼠體內(nèi)成瘤能力,使HEp-2細(xì)胞停滯于G2/M期,最終細(xì)胞凋亡；同時(shí),AURKA沉默后,HEp-2細(xì)胞有絲分裂監(jiān)測點(diǎn)被激活,減少HEp-2細(xì)胞染色體的不穩(wěn)定性；此外,AURKA沉默后,在體外體內(nèi)實(shí)驗(yàn)中,粘著斑激酶(FAK)、磷酸化FAK和基質(zhì)金屬蛋白酶-2(MMP-2)表達(dá)降低；最后,單獨(dú)使用VX-680注射入荷瘤裸鼠,腫瘤生長雖然有所減慢,但是與對照組相比差異無顯著性。 結(jié)論：在喉鱗狀細(xì)胞癌中AURKA呈高表達(dá),與頸部淋巴結(jié)轉(zhuǎn)移和Ⅲ/Ⅳ分期相關(guān),AURKA基因沉默在體外體內(nèi)均能抑制喉癌HEp-2細(xì)胞的生長和侵襲,并與FAK、磷酸化FAK和MMP-2表達(dá)降低有關(guān),本研究為靶向AURKA的喉癌基因治療提供重要的理論依據(jù)。
[Abstract]:Aim: to study the expression and clinical significance of Aurora kinase A Aurora Kinase A (AURKAA) in laryngeal squamous cell carcinoma (LSCC), and to investigate the effect of AURKA gene silencing on the growth of HEp-2 cells in vitro and its mechanism. Methods: 37 cases of laryngeal squamous cell carcinoma and adjacent normal tissues were collected and the expression of AURKA mRNA and protein in laryngeal carcinoma and adjacent normal tissues was detected by real-time fluorescence RT-PCR and Western blot. To analyze the relationship between AURKA mRNA and protein and clinicopathological features of laryngeal carcinoma. ShRNA expression plasmid for AURKA was constructed and transfected into laryngeal squamous cell carcinoma (HEp-2) cell line. The expression of AURKA mRNA and protein in HEp-2 cells before and after transfection were detected by CCK-8 assay, cell proliferation was detected by Transwell assay, and clone formation ability was observed by soft Agar assay. Cell cycle distribution, apoptotic and mitotic monitoring sites were detected by flow cytometry, and intercellular bridges were observed by immunofluorescence assay. Chromosomal instability events, such as multipolar spindle and polycentrosome, were used to detect the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2) protein, which are important factors of cell adhesion and migration. In addition, AURKA knockout HEp-2 cells were inoculated into nude mice. The growth of transplanted tumor and the expression of FAK and MMP-2 in nude mice were observed. Finally, AURKA inhibitor VX-680 was injected into nude mice to observe the effect of VX-680 on tumor growth. Results the expression of AURKA mRNA in tumor of 32 patients with normal laryngeal mucosa was higher than that of normal laryngeal mucosa, and the expression of Aurka mRNA in laryngeal carcinoma was significantly higher than that in normal laryngeal mucosa and normal laryngeal mucosa. The T / N ratio of AURKA protein was 1.2 in 16 patients with laryngeal carcinoma, indicating that the up-regulation of AURKA protein and the high expression of AURKA mRNA were significantly correlated with cervical lymph node metastasis, and also correlated with clinical stage 鈪，

本文編號：1577068