本文關鍵詞： 紫甘藍 花青素 矢車菊-3-O-葡萄糖苷 乳腺癌 黑色素瘤　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

【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,已有研究報道它能抑制癌癥。本研究通過測定幾種常見蔬菜中總花青素含量,發(fā)現(xiàn)紫甘藍為總花青素含量最高的,并且測定了紫甘藍不同部位花青素含量。而紫甘藍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,通過體外體內評價了紫甘藍中花青素和Cy-3-glu對人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),人表皮生長因子受體(HER-2)表達均為陰性的乳腺癌,約占乳腺癌的10-15%,具有遠處轉移風險大,愈后差等特點。黑色素瘤是一種皮膚癌癥,起源于皮膚中的黑素細胞,這兩種癌癥目前都沒有良好的靶向治療方法。本實驗通過單因素實驗考察不同浸提條件對花青素提取效果的影響,選取幾種常見蔬菜用pH差示法測定其總花青素含量,發(fā)現(xiàn)花青素的含量與蔬菜的顏色密切相關：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。所測定的紫色蔬菜中以紫甘藍花青素含量最高。而且同一棵紫甘藍不同部位花青素含量也不同,由外向內呈遞減趨勢。本研究發(fā)現(xiàn)紫甘藍花青素主要成分Cy-3-glu能特異的抑制雌激素受體的變體(ERa36)和表皮生長因子受體(EGFR)共表達的人TNBC細胞系(MDA-MB-231, MDA-MB-436和BT20)生長,并促進其凋亡,而對非TNBC細胞(MCF-7,SK-BR-3)和正常乳腺上皮細胞(MCF-10A)沒有明顯作用。進一步研究發(fā)現(xiàn)Cy-3-glu能直接與NBC細胞膜上的ERα36的配體結合域(LBD)結合抑制其下游EGFR/AKT信號通路并促進EGFR蛋白酶體降解。激活腫瘤壞死因子(TNF-α)誘導激活cleaved-caspase-8/3級聯(lián)凋亡通路,最終不經過線粒體途徑使細胞凋亡。同時,通過給裸鼠腫瘤模型飼喂主要花青素成分為Cy-3-glu的紫甘藍提取物飼料,進一步在小鼠體內驗證體外結果,發(fā)現(xiàn)紫甘藍提取物飼料能顯著抑制腫瘤細胞生長,這與體外結果一致。另外,32例TNBC病人的免疫組化結果表明TNBC病人ERa36(75.00%)與EGFR(68.75%)都高表達,且二者表達有相關性(r=0.536),暗示Cy-3-glu在臨床治療ERα36與EGFR共陽性的TNBC病人潛在的作用。對于黑色素瘤來說,我們發(fā)現(xiàn)Cy-3-glu也能促進小鼠黑色素瘤細胞系(B16-F10)凋亡。進一步研究發(fā)現(xiàn)Cy-3-glu是通過靶向重組酶RAD51使B16-F10細胞周期停滯在G2/M期,破壞同源重組,從而導致細胞凋亡。另外,通過給C57小鼠腫瘤模型飼喂主要花青素成分為Cy-3-glu的紫甘藍提取物飼料,進一步在小鼠體內驗證體外結果,發(fā)現(xiàn)紫甘藍提取物飼料能顯著抑制腫瘤細胞生長,這與體外結果一致。綜上所述,我們發(fā)現(xiàn)紫甘藍中花青素Cy-3-glu通過不同機理抑制癌細胞生長,促進其凋亡。因此我們提出紫甘藍等富含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.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R151
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本文編號：1528605