【摘要】：Notch信號通路作為一條高度保守的信號轉(zhuǎn)導途徑,廣泛表達于從低等的無脊椎動物到高等的哺乳動物中。Notch信號通路對于調(diào)控干細胞的分化、細胞增殖、細胞凋亡等生理活性具有重要的作用。失調(diào)的Notch信號通路在多種人類癌細胞中異常表達,因此Notch信號通路已成為多種惡性腫瘤治療和抗癌藥物篩選的重要靶點之一 黃腐酚(Xanthohumol, XN)作為一種僅存在于啤酒花中的異戊二烯基黃酮類化合物,具有多種生物學活性。黃腐酚在防治糖尿病和動脈粥樣硬化方面起著重要的作用,并且具有抗氧化、抗病毒、抑制乳腺癌、結(jié)腸癌、卵巢癌、前列腺癌等癌細胞生長的作用,然而其發(fā)揮作用的分子機制不詳。鑒于Notch信號通路在前列腺癌細胞中呈現(xiàn)出高度活化的報道,推測黃腐酚抑制前列腺癌細胞的生長可能與調(diào)控細胞中Notch信號通路活性有關(guān)。 本論文旨在研究以Notch信號通路為靶點探討黃腐酚的抗腫瘤活性及其作用機理。首先在前列腺癌細胞PC3中轉(zhuǎn)染基于Notch1的熒光素酶報告質(zhì)粒,構(gòu)建出穩(wěn)轉(zhuǎn)細胞株P(guān)CNL1-2,所得細胞株可以很好地響應(yīng)Notch信號的改變,能夠篩選Notch信號通路抑制劑；通過此細胞株,對實驗室中具有潛在抗癌活性的多種化合物進行篩選,篩選出可以抑制Notch信號通路活性的天然產(chǎn)物黃腐酚；MTT試驗證實濃度為30μmol/L的黃腐酚在12h內(nèi)無細胞毒活性,細胞活性大于80%；而細胞熒光抑制實驗中濃度為30μmol/L的黃腐酚處理穩(wěn)轉(zhuǎn)細胞株P(guān)CNL1-2細胞12h后,細胞熒光的抑制率大于50%,表明黃腐酚可以特異性地抑制Notch信號通路活性；通過流式細胞分析術(shù)與western blot實驗證實黃腐酚可以將細胞周期阻滯在Go/G1期,同時黃腐酚促進了前列腺癌細胞凋亡。通過進一步的細胞轉(zhuǎn)染、western blot、RT-PCR等實驗,對黃腐酚作用的分子靶點進行了探索。研究發(fā)現(xiàn),Notch信號通路與GSK-3p蛋白有著密切的聯(lián)系。GSK-3p可以通過降低Notch蛋白的穩(wěn)定性抑制其信號通路活性,因此可以將GSK-3p看作是Notch信號通路的抑制蛋白之一。本文研究發(fā)現(xiàn)黃腐酚正是通過抑制GSK-3p第9位絲氨酸的磷酸化進而抑制Notch信號通路活性。 綜上所述,黃腐酚通過下調(diào)前列腺癌細胞中高度活化的Notch信號通路活性而抑制前列腺癌細胞的生長。本研究闡述了黃腐酚抗腫瘤活性的分子作用機理,為黃腐酚作為Notch信號通路的抑制劑治療前列腺癌提供了有價值的理論依據(jù)。
[Abstract]:As a highly conserved signal transduction pathway, the Notch signaling pathway is widely expressed in mammals from lower invertebrates to higher mammals. Notch signaling pathway regulates stem cell differentiation and cell proliferation. Apoptosis and other physiological activities play an important role. The dysfunctional Notch signaling pathway is expressed abnormally in a variety of human cancer cells, so the Notch signaling pathway has become one of the important targets for the treatment of many malignant tumors and screening of anticancer drugs (Xanthohumol,). As a kind of isoprene flavonoids only existed in hops, XN has many biological activities. Fulvic phenol plays an important role in the prevention and treatment of diabetes and atherosclerosis, and has anti-oxidation, anti-virus, and inhibit the growth of breast, colon, ovarian, prostate cancer, and other cancer cells. However, the molecular mechanism of its role is unknown. In view of the high activation of Notch signaling pathway in prostate cancer cells, it is suggested that the inhibition of the growth of prostate cancer cells by fulvic phenol may be related to the regulation of the activity of Notch signaling pathway in prostate cancer cells. The aim of this study was to investigate the antitumor activity and mechanism of fulvic phenol by Notch signaling pathway. Firstly, the luciferase reporter plasmid based on Notch1 was transfected into prostate cancer cell line PC3, and the stable cell line PCNL1-2, was constructed, which could respond well to the change of Notch signal and screen the inhibitor of Notch signaling pathway. Through this cell line, many kinds of compounds with potential anticancer activity in the laboratory were screened, and the natural product which could inhibit the activity of Notch signaling pathway was screened out. MTT test showed that 30 渭 mol / L of fulvic phenol had no cytotoxic activity within 12 h, and the cell activity was more than 80%. After treated with 30 渭 mol / L fulvic phenol for 12 h, the fluorescence inhibition rate of PCNL1-2 cells was more than 50%, which indicated that fulvic phenol could specifically inhibit the activity of Notch signaling pathway. Flow cytometry and western blot assay confirmed that fulvic acid could block the cell cycle in Go/G1 phase and promote apoptosis of prostate cancer cells. Further cell transfection, western blot,RT-PCR and other experiments were carried out to explore the molecular targets of the action of fulvic phenol. It has been found that Notch signaling pathway is closely related to GSK-3p protein, and GSK-3p can inhibit its signal pathway activity by reducing the stability of Notch protein, so GSK-3p can be regarded as one of the inhibitory proteins of Notch signaling pathway. In this study, it was found that fulvic phenol inhibited the activity of Notch signaling pathway by inhibiting the phosphorylation of serine at the 9th position of GSK-3p. In conclusion, fulvic phenol inhibits the growth of prostate cancer cells by down-regulating the activity of highly activated Notch signaling pathway in prostate cancer cells. In this study, the molecular mechanism of the antineoplastic activity of fulvic phenol is expounded, which provides a valuable theoretical basis for the treatment of prostate cancer with fulvic phenol as an inhibitor of Notch signaling pathway.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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