自噬在姜黄素诱导的喉癌Hep-2细胞增殖抑制中的作用

发布时间：2017-12-31 21:25

本文关键词：自噬在姜黄素诱导的喉癌Hep-2细胞增殖抑制中的作用　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：姜黄素是姜黄的主要成分,因其抗癌和抗炎的特点而广为人知。研究表明,姜黄素是广谱抗肿瘤药,对多种恶性肿瘤有抑制作用。此外,临床前动物实验和第一阶段临床试验证明姜黄素具有极小的毒性,即使是相对较高的剂量(12 g/天)也没有出现明显不良反应。大多数化疗药物发挥其作用的机制是促进细胞凋亡,而有研究表明自噬在癌症治疗中也发挥作用。姜黄素可以诱导多种肿瘤细胞发生自噬现象,而自噬在姜黄素诱导的喉癌细胞增殖抑制中的作用还未见报道,本研究以人喉癌Hep-2细胞作为研究对象,探究(1)不同浓度的姜黄素对喉癌Hep-2细胞的增殖抑制作用以及对自噬相关蛋白表达的影响;(2)自噬在姜黄素诱导的喉癌细胞凋亡中的作用。研究方法1.MTT法检测不同浓度的姜黄素以及3-MA处理的人喉癌Hep-2细胞的增殖抑制率,体外评价姜黄素以及3-MA对Hep-2的增值抑制作用。2.用一定浓度的姜黄素处理细胞后,Western blots检测细胞自噬相关的蛋白(Beclin1,LC3)表达的变化,以此分析姜黄素对Hep-2细胞自噬的影响。3.使用5m M自噬抑制剂3-甲基腺嘌呤预处理Hep-2细胞2h后,用40μM的姜黄素处理细胞,MTT法检测细胞活力,并与姜黄素单独处理组比较,体外评价姜黄素联合使用自噬抑制剂3-MA后,对Hep-2细胞的增殖抑制作用。4.通过Annexin VFITC/PI流式细胞术检测使用3-MA预处理组以及姜黄素单独处理组对Hep-2细胞的凋亡率。5.荧光染料吖啶橙(AO)染色法定性的观察使用3-MA预处理组以及单独使用姜黄素组细胞自噬水平的变化。6.Western blot检测3-MA预处理组自噬相关蛋白LC3、Beclin1,以及凋亡相关蛋白Bcl-2,Bax的表达水平,并与姜黄素单独处理组以及3-MA单独处理组相比较。研究结果1.MTT结果显示:随着姜黄素以及3-MA浓度的升高以及处理时间的增加,对喉癌Hep-2的抑制作用逐渐增加,呈时间-剂量-依赖性的抑制Hep-2细胞的体外增殖。2.Western blots结果显示,随着姜黄素浓度的升高,自噬相关蛋白Beclin1、LC3Ⅱ的表达量升高,提示细胞自噬水平升高,并具有统计学意义。3.MTT结果显示:使用自噬抑制剂3-MA预处理Hep-2细胞后,再以一定浓度姜黄素处理细胞,与姜黄素单独处理组以及3-MA单独组处理相比,喉癌细胞活力明显下降。4.流式细胞术检测结果显示,使用3-MA预处理细胞组与单独使用姜黄素组相比,细胞凋亡率明显增多。5.AO吖啶橙染色法结果显示,使用3-MA预处理可降低姜黄素引起自噬现象。6.Western blot结果显示使用3-MA预处理喉癌Hep-2细胞后,Bcl-2以及Bclin-1表达水平降低,Bax蛋白表达水平升高,LC3表达减少。结论1.姜黄素能呈时间-剂量-依赖性抑制喉癌Hep-2细胞增殖,同时诱导喉癌Hep-2细胞发生自噬。2.使用自噬抑制剂3-MA抑制自噬后,可增强姜黄素对喉癌Hep-2细胞系的凋亡诱导效应
[Abstract]:Curcumin is the main ingredient of turmeric, known for its characteristics of anticancer and anti-inflammatory. Studies show that curcumin is a broad-spectrum anti-tumor drug, has inhibitory effect on many malignant tumors. In addition, preclinical animal experiments and the first phase of clinical trials proved that curcumin has minimal toxicity, even at relatively high doses (12 g/ day) there is no obvious adverse reaction mechanism. Most chemotherapy drugs play its role is to promote cell apoptosis, and studies have shown that autophagy also plays a role in the treatment of cancer. Curcumin can induce autophagy in tumor cells, and the role of autophagy in laryngeal carcinoma cell proliferation induced by curcumin inhibition has not been reported. In this study, people Hep-2 cancer cells as the research object, to explore (1) inhibition of different concentrations of curcumin on proliferation of human laryngeal carcinoma Hep-2 cells and the related protein expression of autophagy As the influence; (2) the role of autophagy in laryngeal cancer cell apoptosis induced by curcumin in 1.MTT assay. Methods different concentrations of curcumin and the proliferation of 3-MA treated human laryngeal carcinoma Hep-2 cell inhibitory rate was evaluated in vitro inhibition value.2. curcumin and 3-MA on Hep-2 with a certain concentration of curcumin in cells treated by Western blots detection of autophagy related proteins (Beclin1, LC3) expression changes, in order to analyze the effect of curcumin on autophagy in Hep-2 cells using.3. 5m M autophagy inhibitor 3- methyladenine pretreatment Hep-2 2H cells after treatment with curcumin 40 M cells, the cell viability was measured by MTT, and curcumin treatment group, in vitro evaluation of curcumin combined use of autophagy inhibitor 3-MA on the proliferation of Hep-2 cells after inhibition of.4. by Annexin VFITC/PI flow cytometry using 3-MA pretreatment group and ginger Emodin treatment alone group of apoptosis rate of Hep-2 cells.5. fluorescent dye acridine orange (AO) staining qualitative observation using 3-MA pretreatment group and the changes of.6.Western blot using autophagy in curcumin group separate detection of 3-MA pretreatment group of autophagy related protein LC3, Beclin1, and apoptosis related protein Bcl-2, Bax expression, and with curcumin treatment group and 3-MA treatment group compared separately. The results showed that 1.MTT and 3-MA increased with curcumin concentration and treatment time, inhibition on Hep-2 gradually increased, the proliferation of.2.Western blots results showed time dose dependent inhibition of Hep-2 cells showed that with increasing concentration of curcumin the expression of autophagy related protein Beclin1, LC3 II increased, elevated levels of autophagy, and has statistical significance.3.MTT results show: Pretreatment with Hep-2 cell autophagy inhibitor 3-MA, and then to a certain concentration of curcumin treated cells compared with curcumin treatment group and 3-MA alone group,.4. significantly decreased activity of laryngeal carcinoma cells by flow cytometry showed that 3-MA cells pretreated with curcumin group and single group, the apoptosis rate increased significantly.5.AO acridine orange staining results showed that 3-MA pretreatment could reduce the curcumin induced autophagy in.6.Western blot results showed that the pretreatment with 3-MA laryngeal carcinoma cells Hep-2, Bcl-2 and Bclin-1 expression decreased, Bax protein expression increased, LC3 expression decreased. Conclusion curcumin can 1. a time - dose - dependent inhibition of proliferation of Hep-2 cells. At the same time inducing laryngeal cancer Hep-2 cell autophagy.2. using autophagy inhibitor 3-MA inhibiting autophagy, can enhance the effect of curcumin on laryngeal cancer cell line Hep-2. Induction effect

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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