本文關(guān)鍵詞： 紫甘藍(lán) 花青素 矢車菊-3-O-葡萄糖苷 乳腺癌 黑色素瘤　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：花青素(anthocyanin)屬于類黃酮,是植物中的一種水溶性天然色素,在蔬菜中分布極廣,已有研究報(bào)道它能抑制癌癥。本研究通過(guò)測(cè)定幾種常見(jiàn)蔬菜中總花青素含量,發(fā)現(xiàn)紫甘藍(lán)為總花青素含量最高的,并且測(cè)定了紫甘藍(lán)不同部位花青素含量。而紫甘藍(lán)中主要的花青素成分是矢車菊-3-O-葡萄糖苷(Cy-3-glu)。所以本研究利用人三陰性乳腺癌(TNBC)和小鼠黑色素瘤為模型,通過(guò)體外體內(nèi)評(píng)價(jià)了紫甘藍(lán)中花青素和Cy-3-glu對(duì)人TNBC和小鼠黑色素瘤的作用。TNBC是指雌激素受體(ER),孕激素受體(PR),人表皮生長(zhǎng)因子受體(HER-2)表達(dá)均為陰性的乳腺癌,約占乳腺癌的10-15%,具有遠(yuǎn)處轉(zhuǎn)移風(fēng)險(xiǎn)大,愈后差等特點(diǎn)。黑色素瘤是一種皮膚癌癥,起源于皮膚中的黑素細(xì)胞,這兩種癌癥目前都沒(méi)有良好的靶向治療方法。本實(shí)驗(yàn)通過(guò)單因素實(shí)驗(yàn)考察不同浸提條件對(duì)花青素提取效果的影響,選取幾種常見(jiàn)蔬菜用pH差示法測(cè)定其總花青素含量,發(fā)現(xiàn)花青素的含量與蔬菜的顏色密切相關(guān)：紫色蔬菜比紅色蔬菜花青素含量高,紅色蔬菜比黃色蔬菜花青素含量高。所測(cè)定的紫色蔬菜中以紫甘藍(lán)花青素含量最高。而且同一棵紫甘藍(lán)不同部位花青素含量也不同,由外向內(nèi)呈遞減趨勢(shì)。本研究發(fā)現(xiàn)紫甘藍(lán)花青素主要成分Cy-3-glu能特異的抑制雌激素受體的變體(ERa36)和表皮生長(zhǎng)因子受體(EGFR)共表達(dá)的人TNBC細(xì)胞系(MDA-MB-231, MDA-MB-436和BT20)生長(zhǎng),并促進(jìn)其凋亡,而對(duì)非TNBC細(xì)胞(MCF-7,SK-BR-3)和正常乳腺上皮細(xì)胞(MCF-10A)沒(méi)有明顯作用。進(jìn)一步研究發(fā)現(xiàn)Cy-3-glu能直接與NBC細(xì)胞膜上的ERα36的配體結(jié)合域(LBD)結(jié)合抑制其下游EGFR/AKT信號(hào)通路并促進(jìn)EGFR蛋白酶體降解。激活腫瘤壞死因子(TNF-α)誘導(dǎo)激活cleaved-caspase-8/3級(jí)聯(lián)凋亡通路,最終不經(jīng)過(guò)線粒體途徑使細(xì)胞凋亡。同時(shí),通過(guò)給裸鼠腫瘤模型飼喂主要花青素成分為Cy-3-glu的紫甘藍(lán)提取物飼料,進(jìn)一步在小鼠體內(nèi)驗(yàn)證體外結(jié)果,發(fā)現(xiàn)紫甘藍(lán)提取物飼料能顯著抑制腫瘤細(xì)胞生長(zhǎng),這與體外結(jié)果一致。另外,32例TNBC病人的免疫組化結(jié)果表明TNBC病人ERa36(75.00%)與EGFR(68.75%)都高表達(dá),且二者表達(dá)有相關(guān)性(r=0.536),暗示Cy-3-glu在臨床治療ERα36與EGFR共陽(yáng)性的TNBC病人潛在的作用。對(duì)于黑色素瘤來(lái)說(shuō),我們發(fā)現(xiàn)Cy-3-glu也能促進(jìn)小鼠黑色素瘤細(xì)胞系(B16-F10)凋亡。進(jìn)一步研究發(fā)現(xiàn)Cy-3-glu是通過(guò)靶向重組酶RAD51使B16-F10細(xì)胞周期停滯在G2/M期,破壞同源重組,從而導(dǎo)致細(xì)胞凋亡。另外,通過(guò)給C57小鼠腫瘤模型飼喂主要花青素成分為Cy-3-glu的紫甘藍(lán)提取物飼料,進(jìn)一步在小鼠體內(nèi)驗(yàn)證體外結(jié)果,發(fā)現(xiàn)紫甘藍(lán)提取物飼料能顯著抑制腫瘤細(xì)胞生長(zhǎng),這與體外結(jié)果一致。綜上所述,我們發(fā)現(xiàn)紫甘藍(lán)中花青素Cy-3-glu通過(guò)不同機(jī)理抑制癌細(xì)胞生長(zhǎng),促進(jìn)其凋亡。因此我們提出紫甘藍(lán)等富含Cy-3-glu的果蔬將有望成為臨床治療或輔助治療TNBC和黑色素瘤的潛在食療產(chǎn)品。
[Abstract]:Anthocyanin, a kind of flavonoid, is a kind of water-soluble natural pigment in plants, which is widely distributed in vegetables. It has been reported that anthocyanin can inhibit cancer. It was found that purple cabbage had the highest total anthocyanin content. The anthocyanin content in different parts of purple cabbage was determined, and the main anthocyanin component in purple cabbage was Cy-3-gluonine. So the study used human three-negative breast cancer TNBC) and mouse melanoma as the model. The effects of anthocyanin and Cy-3-glu on human TNBC and mouse melanoma were evaluated in vitro. About 10-15 percent of breast cancer is characterized by a high risk of distant metastasis and poor recovery. Melanoma is a skin cancer that originates from melanocytes in the skin. There is no good target therapy for these two kinds of cancer at present. The effects of different extraction conditions on anthocyanin extraction were investigated by single factor experiment, and some common vegetables were selected to determine the total anthocyanin content by pH difference method. It was found that the content of anthocyanin was closely related to the color of vegetables: purple vegetables had higher anthocyanin content than red vegetables. The anthocyanin content of red vegetables is higher than that of yellow vegetables, and the anthocyanin content of purple vegetables is the highest, and the anthocyanin content of different parts of the same purple cabbage is different. In this study, we found that Cy-3-glu, the main component of anthocyanin, can specifically inhibit the growth and apoptosis of human TNBC cell line MDA-MB-231, MDA-MB-436 and BT20, which can specifically inhibit the expression of estrogen receptor variant ERa36) and epidermal growth factor receptor (EGFR). There was no significant effect on MCF-7 SK-BR-3) and normal breast epithelial cells (MCF-10A). Further studies showed that Cy-3-glu could directly bind to the ligand binding domain of ER 偽 36 on NBC cell membrane and inhibit its downstream EGFR/AKT signaling pathway and promote EGFR proteasome. Degradation. Activation of tumor necrosis factor TNF- 偽 (TNF- 偽) induces activation of cleaved-caspase-8/3 cascade apoptosis pathway, At the same time, the tumor model of nude mice was fed with Violet Cabbage extract (Cy-3-glu), which was the main component of anthocyanin, to further verify the results in vivo and in vitro. In addition, the immunohistochemical results of 32 patients with TNBC showed that ERa3675.00 and EGFR 68.75) were highly expressed. There was a correlation between the two expressions, suggesting the potential role of Cy-3-glu in the clinical treatment of TNBC patients with co-positive ER 偽 36 and EGFR. We found that Cy-3-glu could also promote the apoptosis of mouse melanoma cell line B16-F10. Further studies showed that Cy-3-glu caused the cell cycle arrest in G _ 2 / M phase by targeting the recombinant enzyme RAD51, which destroyed homologous recombination and led to apoptosis. By feeding C57 mice tumor model with Violet cabbage extract feed with Cy-3-glu as the main anthocyanin component, the results were further verified in vivo and in vitro. It was found that the extract feed could significantly inhibit the growth of tumor cells. In conclusion, we found that anthocyanin Cy-3-glu in purple cabbage inhibits the growth of cancer cells through different mechanisms. Therefore, we suggest that fruits and vegetables rich in Cy-3-glu, such as purple cabbage, may become potential therapeutic products for TNBC and melanoma in clinical treatment or adjuvant therapy.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R151
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本文編號(hào)：1528605