香芹酚对非小细胞肺癌1299细胞功能影响的实验研究

发布时间：2018-04-21 00:32

本文选题：香芹酚 + 人非小细胞肺癌NCI-H1299细胞　；参考：《广西医科大学》2017年硕士论文

【摘要】：背景与目的:肺癌是全世界癌症相关死亡的主要原因,因缺乏有效的治疗导致高发病率和死亡率,每年造成约150万人死亡。肺癌分为非小细胞肺癌(non-small cell lung cancer,NSCLC)和小细胞肺癌(small cell lung cancer,SCLC),其中NSCLC占所有肺癌的85%,约75%的NSCLC患者发现时已处于中晚。尽管基础和临床研究的进步,但非小细胞肺癌的总体5年生存率仅为15%。NSCLC的发生与发展受多种因素影响,表现为多阶段的复杂过程,发病机制至今尚未清楚。因此,探索肺癌发生、恶化和进展的机制,寻找有效的诊断和治疗靶标是必要的。从植物及中草药中提取有效且毒副反应小的成分作用于相应靶点已经成为国内外学者研发抗肿瘤药物的新途径。香芹酚(carvacrol,CV),化学结构为5-异丙基-2-甲基苯酚,是一种单帖酚,从许多植物中提取,包括水果,蔬菜,香料和草药,香芹酚具有多种用途,如作为安全的添加剂用于化妆品和食品的生产过程中,是百里香油、香薰精油的主要成分。香芹酚已证实具有多方向的药理作用。多项研究表明,CV具有多种生物学功能,如抗炎症反应、抗氧化应激及抗肿瘤等,且对人机体组织细胞毒性较小。近年研究发现,香芹酚能抑制多种肿瘤细胞的生长,如肝癌细胞、转移性乳腺癌细胞等,香芹酚对多种肿瘤细胞的具有抑制增殖及诱导凋亡作用,然而香芹酚抗肺癌的机制尚未阐明。目前研究表明香芹酚对非小细胞肺癌A549有显著促进凋亡的作用,但作用机制尚无报道。A549及NCI-H1299同属非小细胞肺癌细胞系,但NCI-H1299基因组DNA部分P53缺失,不能表达P53蛋白,故两者的凋亡水平及机制有所区别,对抗癌治疗的敏感性及有效性亦存在差异。香芹酚针对NCI-H1299细胞功能的影响,国内外尚未见文献报道。在此基础上,我们将CV作用于非小细胞肺癌NCI-H1299细胞,选择含半胱氨酸的天冬氨酸蛋白水解酶-9(caspase-9)、基质金属蛋白酶-9(MMP-9)及其抑制剂(TIMP-1)为作用靶点,观察CV对肿瘤细胞凋亡侵袭作用的影响,并初步探讨相关机制,为非小细胞肺癌的精准治疗提供新的策略。方法:以不同浓度的香芹酚(0.00、20.0、40.0、60.0、80.0μmol/L)处理NCI-H1299细胞后,采用四甲基偶氮唑蓝(MTT)比色法检测细胞活力;Transwell法检测细胞侵袭能力;流式细胞仪(FCM)技术检测细胞凋亡;Quantitative Realtime-PCR及Western blot检测caspase-9、MMP-9、TIMP-1在核酸及蛋白水平的变化。结果:香芹酚显著抑制NCI-H1299细胞增殖,不表现为浓度依赖性及时间依赖性(P0.05);有效诱导NCI-H1299细胞凋亡(P0.05),不表现为浓度依赖性(P0.05);明显抑制NCI-H1299细胞侵袭(P0.05)。进一步的QT-PCR及Western blot实验表明,香芹酚选择性激活Caspase-9,同时MMP-9表达受到抑制而TIMP-1表达升高(P0.05)。结论:香芹酚可通过激活Caspase-9诱导人非小细胞肺癌NCI-H1299细胞凋亡,通过下调MMP-9抑制细胞侵袭。
[Abstract]:Background & objective: lung cancer is the leading cause of cancer related deaths worldwide, resulting in high morbidity and mortality due to the lack of effective treatment, resulting in about 1.5 million deaths each year. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), in which NSCLC accounts for 85% of all lung cancers. About 75% of NSCLC patients were found in the middle and late stages. Despite the progress of basic and clinical studies, the overall 5-year survival rate of non-small cell lung cancer is affected by many factors, such as the occurrence and development of 15%.NSCLC. Therefore, it is necessary to explore the mechanism of occurrence, deterioration and progression of lung cancer and to find effective diagnostic and therapeutic targets. It has become a new way for domestic and foreign scholars to develop antitumor drugs by extracting effective and small toxic and side effect components from plants and Chinese herbal medicines. Carvacrolan, a chemical structure of 5-isopropyl-2-methylphenol, is a monophenol extracted from many plants, including fruits, vegetables, spices and herbs. As a safe additive used in cosmetics and food production, thyme oil, aromatherapy essential oil is the main ingredient. Carvingol has been proved to have multi-directional pharmacological effects. Many studies have shown that CV has a variety of biological functions, such as anti-inflammatory response, anti-oxidative stress and anti-tumor, and has less cytotoxicity to human tissues and cells. In recent years, it has been found that carvingol can inhibit the growth of many kinds of tumor cells, such as hepatoma cells, metastatic breast cancer cells and so on. Carvingol can inhibit the proliferation and induce apoptosis of many kinds of tumor cells. However, the mechanism of carvingol against lung cancer has not been elucidated. Recent studies have shown that carvonol can significantly promote apoptosis in non-small cell lung cancer (NSCLC) A549. However, there is no report on the mechanism. A549 and NCI-H1299 belong to NSCLC cell line, but NCI-H1299 genome DNA is partially p53 deleted and can not express p53 protein. Therefore, the level and mechanism of apoptosis were different, and the sensitivity and effectiveness of anticancer therapy were also different. The effect of carvingol on the function of NCI-H1299 cells has not been reported in the literature at home and abroad. On this basis, CV was applied to NCI-H1299 cells of non-small cell lung cancer. The target was aspartate proteolytic enzyme -9caspase-9, matrix metalloproteinase-9 (MMP-9) and its inhibitor, TIMP-1, which contained cysteine. To observe the effect of CV on tumor cell apoptosis and invasion, and to explore the related mechanism, and to provide a new strategy for accurate treatment of non-small cell lung cancer (NSCLC). Methods: NCI-H1299 cells were treated with different concentrations of carvilinol (0.0020. 0) and 40.060. 0. 0 渭 mol / L respectively. The invasion ability of NCI-H1299 cells was detected by MTT colorimetry. Quantitative Realtime-PCR and Western blot were used to detect the changes of caspase-9 MMP-9 and TIMP-1 in nucleic acid and protein levels. Results: carvonol significantly inhibited the proliferation of NCI-H1299 cells, not in a concentration-dependent or time-dependent manner, but not in a dose-dependent manner, but in a dose-dependent manner. It also inhibited the invasion of NCI-H1299 cells by P0.05, and induced the apoptosis of NCI-H1299 cells in a dose-dependent manner. Further QT-PCR and Western blot experiments showed that carvophanol selectively activated Caspase-9, while the expression of MMP-9 was inhibited and the expression of TIMP-1 increased (P 0.05). Conclusion: carvingol can induce apoptosis of NCI-H1299 cells by activating Caspase-9, and inhibit the invasion of NCI-H1299 cells by down-regulating MMP-9.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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