本文選題：香芹酚 + 人非小細(xì)胞肺癌NCI-H1299細(xì)胞　； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景與目的:肺癌是全世界癌癥相關(guān)死亡的主要原因,因缺乏有效的治療導(dǎo)致高發(fā)病率和死亡率,每年造成約150萬人死亡。肺癌分為非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)和小細(xì)胞肺癌(small cell lung cancer,SCLC),其中NSCLC占所有肺癌的85%,約75%的NSCLC患者發(fā)現(xiàn)時(shí)已處于中晚。盡管基礎(chǔ)和臨床研究的進(jìn)步,但非小細(xì)胞肺癌的總體5年生存率僅為15%。NSCLC的發(fā)生與發(fā)展受多種因素影響,表現(xiàn)為多階段的復(fù)雜過程,發(fā)病機(jī)制至今尚未清楚。因此,探索肺癌發(fā)生、惡化和進(jìn)展的機(jī)制,尋找有效的診斷和治療靶標(biāo)是必要的。從植物及中草藥中提取有效且毒副反應(yīng)小的成分作用于相應(yīng)靶點(diǎn)已經(jīng)成為國(guó)內(nèi)外學(xué)者研發(fā)抗腫瘤藥物的新途徑。香芹酚(carvacrol,CV),化學(xué)結(jié)構(gòu)為5-異丙基-2-甲基苯酚,是一種單帖酚,從許多植物中提取,包括水果,蔬菜,香料和草藥,香芹酚具有多種用途,如作為安全的添加劑用于化妝品和食品的生產(chǎn)過程中,是百里香油、香薰精油的主要成分。香芹酚已證實(shí)具有多方向的藥理作用。多項(xiàng)研究表明,CV具有多種生物學(xué)功能,如抗炎癥反應(yīng)、抗氧化應(yīng)激及抗腫瘤等,且對(duì)人機(jī)體組織細(xì)胞毒性較小。近年研究發(fā)現(xiàn),香芹酚能抑制多種腫瘤細(xì)胞的生長(zhǎng),如肝癌細(xì)胞、轉(zhuǎn)移性乳腺癌細(xì)胞等,香芹酚對(duì)多種腫瘤細(xì)胞的具有抑制增殖及誘導(dǎo)凋亡作用,然而香芹酚抗肺癌的機(jī)制尚未闡明。目前研究表明香芹酚對(duì)非小細(xì)胞肺癌A549有顯著促進(jìn)凋亡的作用,但作用機(jī)制尚無報(bào)道。A549及NCI-H1299同屬非小細(xì)胞肺癌細(xì)胞系,但NCI-H1299基因組DNA部分P53缺失,不能表達(dá)P53蛋白,故兩者的凋亡水平及機(jī)制有所區(qū)別,對(duì)抗癌治療的敏感性及有效性亦存在差異。香芹酚針對(duì)NCI-H1299細(xì)胞功能的影響,國(guó)內(nèi)外尚未見文獻(xiàn)報(bào)道。在此基礎(chǔ)上,我們將CV作用于非小細(xì)胞肺癌NCI-H1299細(xì)胞,選擇含半胱氨酸的天冬氨酸蛋白水解酶-9(caspase-9)、基質(zhì)金屬蛋白酶-9(MMP-9)及其抑制劑(TIMP-1)為作用靶點(diǎn),觀察CV對(duì)腫瘤細(xì)胞凋亡侵襲作用的影響,并初步探討相關(guān)機(jī)制,為非小細(xì)胞肺癌的精準(zhǔn)治療提供新的策略。方法:以不同濃度的香芹酚(0.00、20.0、40.0、60.0、80.0μmol/L)處理NCI-H1299細(xì)胞后,采用四甲基偶氮唑藍(lán)(MTT)比色法檢測(cè)細(xì)胞活力;Transwell法檢測(cè)細(xì)胞侵襲能力;流式細(xì)胞儀(FCM)技術(shù)檢測(cè)細(xì)胞凋亡;Quantitative Realtime-PCR及Western blot檢測(cè)caspase-9、MMP-9、TIMP-1在核酸及蛋白水平的變化。結(jié)果:香芹酚顯著抑制NCI-H1299細(xì)胞增殖,不表現(xiàn)為濃度依賴性及時(shí)間依賴性(P0.05);有效誘導(dǎo)NCI-H1299細(xì)胞凋亡(P0.05),不表現(xiàn)為濃度依賴性(P0.05);明顯抑制NCI-H1299細(xì)胞侵襲(P0.05)。進(jìn)一步的QT-PCR及Western blot實(shí)驗(yàn)表明,香芹酚選擇性激活Caspase-9,同時(shí)MMP-9表達(dá)受到抑制而TIMP-1表達(dá)升高(P0.05)。結(jié)論:香芹酚可通過激活Caspase-9誘導(dǎo)人非小細(xì)胞肺癌NCI-H1299細(xì)胞凋亡,通過下調(diào)MMP-9抑制細(xì)胞侵襲。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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