【摘要】：目前肺癌仍是世界范圍內(nèi)一種嚴(yán)重威脅人類生命的腫瘤疾病,并且在我國該病的發(fā)病率有逐漸升高的趨勢。盡管針對(duì)肺癌有許多治療方法,但患者的5年存活率依然低于15%,且藥物具有巨大的毒副作用,因此研發(fā)具有低毒高效抗腫瘤藥物的需求仍是迫切的。木香烴內(nèi)酯是從菊科木香屬類植物中提取的一類倍半萜類化合物,能夠體外抑制多種腫瘤細(xì)胞的生長,但對(duì)肺癌細(xì)胞的作用未見報(bào)道。本論文首先采用體外細(xì)胞生長抑制試驗(yàn)(MTT)檢測木香烴內(nèi)酯對(duì)不同腫瘤細(xì)胞生長抑制的作用,從而篩選出對(duì)藥物最敏感的A549細(xì)胞；進(jìn)而以A549細(xì)胞為研究對(duì)象,采用流式細(xì)胞術(shù)觀察藥物處理后細(xì)胞內(nèi)ROS水平、膜電位以及細(xì)胞凋亡變化情況；此外還用激光共聚焦顯微鏡對(duì)細(xì)胞內(nèi)鈣離子以及膜電位變化情況進(jìn)行觀察；western blot (蛋白免疫印跡法)對(duì)細(xì)胞內(nèi)內(nèi)質(zhì)網(wǎng)應(yīng)激以及凋亡相關(guān)蛋白表達(dá)水平進(jìn)行檢測。具體得到的結(jié)果如下：1、MTT結(jié)果表明,木香烴內(nèi)酯能體外抑制A549、HepG2、Hela細(xì)胞生長,且對(duì)肺癌細(xì)胞的生長抑制作用最明顯,并且這種抑制作用表現(xiàn)一定的濃度和時(shí)間梯度依賴。2、以A549細(xì)胞為模型,應(yīng)用流式細(xì)胞技術(shù),通過Annexin V/PI雙染細(xì)胞后觀察不同濃度的木香烴內(nèi)酯處理細(xì)胞24h的凋亡情況。結(jié)果表明,細(xì)胞凋亡率隨著藥物濃度的升高而增加,呈現(xiàn)出一定的濃度梯度依賴性。3、木香烴內(nèi)酯處理A549細(xì)胞后發(fā)現(xiàn),能夠顯著提升細(xì)胞內(nèi)ROS的水平,并誘導(dǎo)內(nèi)質(zhì)網(wǎng)發(fā)生應(yīng)激,內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白GRP78, IRE1α, CHOP等蛋白水平隨著木香烴內(nèi)酯濃度的升高而增加,這一過程伴隨著胞質(zhì)中鈣離子水平的升高。通過RNA干擾技術(shù)抑制IRE1α表達(dá)可以使木香烴內(nèi)酯誘導(dǎo)的細(xì)胞凋亡率下降,證明了內(nèi)質(zhì)網(wǎng)應(yīng)激在木香烴內(nèi)酯誘導(dǎo)凋亡中的作用。進(jìn)一步研究表明通過IRE1α-ASK1-JNK通路磷酸化Bcl-2蛋白,使其失去抗凋亡作用,改變了線粒體膜通透性。應(yīng)用流式細(xì)胞技術(shù),通過JC-1染色發(fā)現(xiàn)藥物處理后線粒體膜電位顯著降低�？寡趸瘎㎞AC能夠恢復(fù)細(xì)胞活性、線粒體膜電位以及有效抑制細(xì)胞凋亡的發(fā)生。表明ROS是木香烴內(nèi)酯誘導(dǎo)A549細(xì)胞發(fā)生凋亡的上游信號(hào)。4、不同的藥物濃度處理A549細(xì)胞后對(duì)caspase活性進(jìn)行檢測,結(jié)果表明木香烴內(nèi)酯能夠激活caspase3和caspase9。caspase抑制劑Z-VAD-FMK能夠有效恢復(fù)細(xì)胞生長活性；western blot檢測顯示,木香烴內(nèi)酯上調(diào)了Bax、下調(diào)了Bcl-2的表達(dá)量,并且促使細(xì)胞色素c從線粒體釋放,激活了caspase3,最終切割PARP,導(dǎo)致細(xì)胞發(fā)生線粒體途徑的凋亡。本文首次闡明木香烴內(nèi)酯通過內(nèi)質(zhì)網(wǎng)氧化應(yīng)激介導(dǎo)IRE1α-JNK的激活,導(dǎo)致Bcl-2磷酸化失去抗凋亡作用,誘導(dǎo)A549細(xì)胞發(fā)生線粒體途徑凋亡。研究結(jié)果進(jìn)一步揭示木香烴內(nèi)酯抗腫瘤機(jī)理,為將來木香烴內(nèi)酯能夠作為臨床新型抗腫瘤藥物提供相關(guān)的理論依據(jù)
[Abstract]:At present, lung cancer is still a serious threat to human life in the world, and the incidence of lung cancer is gradually increasing in China. Although there are many treatments for lung cancer, the 5-year survival rate of patients is still lower than 15%, and the drug has great toxicity and side effects, so it is urgent to develop anti-tumor drugs with low toxicity and high efficiency. Xylenolactone is a kind of sesquiterpenoids extracted from Compositae, which can inhibit the growth of many kinds of tumor cells in vitro, but the effect on lung cancer cells has not been reported. In this paper, in vitro cell growth inhibition test (MTT) was used to detect the inhibitory effect of xylinolactone on different tumor cells, so as to select the most sensitive A549 cells. The changes of ROS level, membrane potential and apoptosis of A549 cells were observed by flow cytometry. The changes of intracellular Ca ~ (2 +) and membrane potential were observed by laser confocal microscopy (LSCM). The changes of intracellular endoplasmic reticulum (ER) stress and the expression of apoptosis-related proteins were detected by; western blot (Western blot. The results were as follows: 1 MTT results showed that xylinolactone could inhibit the growth of A549 HepG2Hela cells in vitro, and had the most obvious inhibitory effect on the growth of lung cancer cells. The inhibition showed a certain concentration and time gradient dependence. 2. A549 cells were used as a model. Flow cytometry was used to observe the apoptosis of A549 cells treated with different concentrations of xylinolactone for 24 hours by Annexin V/PI double staining. The results showed that the apoptosis rate of A549 cells increased with the increase of drug concentration, and showed a certain concentration gradient dependence. 3. After treated with xylinolactone, it was found that the level of ROS in A549 cells could be significantly increased by xylinolactone. Endoplasmic reticulum stress proteins (GRP78, IRE1 偽, CHOP, etc.) increased with the increase of xylinolactone concentration, which was accompanied by the increase of calcium level in the cytoplasm. Inhibition of IRE1 偽 expression by RNA interference technique can decrease the apoptosis rate induced by xylinolactone, which proves the role of endoplasmic reticulum stress in the apoptosis induced by xylinolactone. Further studies showed that the phosphorylated Bcl-2 protein was phosphorylated through the IRE1 偽-ASK1-JNK pathway, which caused it to lose its anti-apoptotic effect and change the permeability of mitochondrial membrane. Flow cytometry and JC-1 staining showed that mitochondrial membrane potential was significantly decreased after drug treatment. Antioxidant NAC can restore cell activity, mitochondrial membrane potential and inhibit apoptosis. The results showed that ROS was the upstream signal of A549 cells apoptosis induced by xylinolactone. 4. The activity of caspase was detected after different concentrations of drugs were used to treat A549 cells. The results showed that xylinolactone could activate caspase3 and caspase9.caspase inhibitor Z-VAD-FMK could effectively restore cell growth activity. Western blot analysis showed that xylinolactone upregulated Bax, down-regulated the expression of Bcl-2 and promoted the release of cytochrome c from mitochondria, which activated caspase3, to cut PARP, and lead to apoptosis of mitochondria pathway. It is the first time to elucidate the activation of IRE1 偽-JNK mediated by xylinolactone through endoplasmic reticulum oxidative stress, which results in Bcl-2 phosphorylation losing its antiapoptotic effect and inducing apoptosis of mitochondria pathway in A549 cells. The results further revealed the antitumor mechanism of xylenolactone, and provided the relevant theoretical basis for the future use of xylol lactone as a new clinical antitumor drug.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王曉慶,梁中琴,顧振綸;姜黃素抗腫瘤作用機(jī)制研究進(jìn)展[J];中草藥;2004年03期

2 Eckart Laack,Dieter Kurt Hossfeld,廖永德;非小細(xì)胞性肺癌的治療[J];The Chinese-German Journal of Clinical Oncology;2000年06期

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本文編號(hào)：2419784