【摘要】：研究背景:乳腺癌是危害女性健康的常見惡性腫瘤之一,是城市女性最常見的癌癥,且發(fā)病率和死亡率逐年上升。乳腺癌是一種多因素,多基因相互作用引起的癌癥,但病因及發(fā)病機制尚未完全清楚。目前乳腺癌的治療雖已逐漸從簡單的手術、化療轉(zhuǎn)向手術、化療、放療以及生物靶向治療等多種手段綜合治療的方向,但仍會對患者身心造成巨大的創(chuàng)傷。因此,尋找高效、低副作用的天然化學藥物對乳腺癌的預防和治療具有重大意義。葉黃素(Lutein)又稱為植物黃體素,是一種具有抗氧化作用的天然植物化合物,大量存在于花卉、蔬果等綠色植物中,尤其在萬壽菊中含量最為豐富。人們通過膳食攝取的葉黃素可經(jīng)由血液輸送到各組織。流行病學研究顯示,葉黃素在保護視力、預防白內(nèi)障、延緩動脈粥樣硬化和預防腫瘤等多方面具有重要作用。目的:本實驗旨在通過檢測葉黃素(Lutein)對人乳腺癌MCF-7細胞中激活蛋白1(activator protein 1,AP-1)及其上下游信號通路中相關因子的影響,進而研究葉黃素對人乳腺癌MCF-7細胞增殖的影響及其作用機制,為乳腺癌的化學預防及葉黃素作為低副作用的天然抗癌藥物的臨床研究提供理論依據(jù)。方法:1.MTT法檢測不同濃度葉黃素(Lutein)對人乳腺癌MCF-7細胞的增殖抑制率。用不同濃度的葉黃素(0、5、10、20、40、80μg/ml)分別處理MCF-7細胞12、24、48h后,用MTT法檢測葉黃素對細胞增殖的抑制率。2.流式細胞術檢測不同濃度的葉黃素(0、5、10、20、40、80μg/ml)分別處理MCF-7細胞24h后的活性氧(reactive oxygen species,ROS)水平。3.采用RT-q PCR檢測各組細胞中Nrf2、HO-1的m RNA的表達水平。4.采用Western blot檢測各組細胞胞漿和細胞核中Nrf2和HO-1的蛋白表達水平。5.根據(jù)以上實驗結果分析設置空白組、3-MA組、葉黃素組、3-MA+葉黃素組進行后續(xù)實驗。流式細胞術檢測空白組及各實驗組細胞周期的變化及凋亡率。6.采用RT-q PCR檢測空白組及各實驗組細胞中PI3K、AP-1、Cox-2和Cyclin D1的m RNA表達水平。7.采用Western blot檢測空白組及各實驗組細胞中PI3K、AP-1、Cox-2和Cyclin D1的蛋白表達水平。結果1.MTT檢測結果顯示,葉黃素能夠抑制人乳腺癌MCF-7細胞的增殖,且抑制作用具有時間和劑量依賴性,即作用時間越長,用藥濃度越大時,對MCF-7細胞的增殖抑制作用越強。2.流式細胞術檢測結果顯示,各組細胞中ROS水平有差異,且隨著葉黃素濃度的增大,ROS水平降低更顯著。3.RT-qPCR的實驗結果顯示,與空白組相比,各濃度組Nrf2、HO-1的mRNA表達水平均上調(diào)。4.Western blot的實驗結果顯示,與空白組相比,HO-1的蛋白表達量呈上調(diào)趨勢,細胞胞漿中Nrf2的蛋白含量基本無差異,而核Nrf2蛋白的含量明顯上調(diào)。5.Annexin V-FITC/PI細胞凋亡檢測結果顯示,24h的3-MA組、葉黃素組(40μg/ml)和3-MA+葉黃素(40μg/ml)組的細胞凋亡率與空白組相比均有明顯差異(P0.05),3-MA+葉黃素(40μg/ml)組細胞凋亡率明顯大于葉黃素(40μg/ml)組和3-MA組(P0.05),而葉黃素(40μg/ml)組與3-MA組相比,兩組無明顯差異。6.細胞周期檢測結果顯示,與空白組相比,各實驗組G0/G1期細胞明顯增加,S期細胞明顯減少,且3-MA+葉黃素組更顯著,表現(xiàn)為顯著地G1期阻滯(P㩳0.05)。7.RT-q PCR的實驗結果顯示,PI3K、AP-1、Cox-2、Cyclin D1的m RNA表達水平各實驗組與空白組相比均下調(diào);葉黃素(40μg/ml)組和3-MA組相比差異較小,3-MA+葉黃素(40μg/ml)組下調(diào)更顯著(P0.05)。8.Western blot的實驗結果顯示,PI3K、AP-1、Cox-2、Cyclin D1的蛋白表達水平各實驗組與空白組相比均下調(diào);葉黃素(40μg/ml)組和3-MA組相比差異較小,3-MA+葉黃素(40μg/ml)組下調(diào)相對更顯著(P0.05)。結論1.葉黃素可明顯抑制人乳腺癌MCF-7細胞的增殖,抑制作用具有時間和劑量依賴性。2.葉黃素可誘導Nrf2的表達和核轉(zhuǎn)位,促進抗氧化酶HO-1的表達,進而降低氧化應激水平。3.葉黃素對人乳腺癌MCF-7細胞增值抑制作用可能是通過上調(diào)人乳腺癌細胞內(nèi)Nrf2的表達,從而促進HO-1的表達,降低細胞內(nèi)的ROS含量,抑制PI3K的表達,進而導致AP-1及其下游靶基因的的表達下調(diào)所介導的。
[Abstract]:Background: Breast cancer is one of the most common malignant tumors that are harmful to women's health. It is the most common cancer in urban women, and the morbidity and mortality are increasing year by year. Breast cancer is a multi-factor, multi-gene-induced cancer, but the cause and pathogenesis of breast cancer have not been completely clear. The current treatment of breast cancer has gradually changed from simple operation, chemotherapy to operation, chemotherapy, radiotherapy and biological targeted therapy, but still causes great trauma to the patient's body and mind. Therefore, it is of great significance to find the natural chemical medicine with high efficiency and low side effect to the prevention and treatment of breast cancer. Lutein is also called a plant yellow voxel, is a natural plant compound with anti-oxidation effect, and is widely used in green plants such as flowers, fruits and vegetables, especially in marigold. Lutein, which is ingested by the diet, can be delivered to the tissues via the blood. The epidemiological study shows that lutein plays an important role in protecting vision, preventing cataract, delaying atherosclerosis and preventing tumor. Objective: To study the effect of lutein on the activation protein 1 (AP-1) in human breast cancer MCF-7 cells and its related factors in the upstream and downstream signal pathways, and to study the effect of lutein on the proliferation of human breast cancer MCF-7 cells and its mechanism of action. provides a theoretical basis for the clinical research of the chemical prevention and the lutein as the low-side effect of the breast cancer. Methods: 1. MTT method was used to detect the inhibitory rate of Lutein on the proliferation of human breast cancer MCF-7 cells. The cells of MCF-7 were treated with different concentrations of lutein (0, 5, 10, 20, 40, 80 & mu; g/ ml), respectively. Lutein (0, 5, 10, 20, 40, 80. mu.g/ ml) of different concentrations were detected by flow cytometry to treat the level of reactive oxygen species (ROS) after 24 h of MCF-7 cells, respectively. The expression level of Nrf2 and HO-1 in each group was detected by RT-q PCR. The expression levels of Nrf2 and HO-1 in the cytoplasm and nucleus of each group were detected by Western blot. The following experiments were carried out in the blank group, 3-MA group, lutein group and 3-MA + lutein group according to the above experimental results. The changes of the cell cycle and the apoptosis rate of the blank group and each experimental group were detected by flow cytometry. The mRNA expression levels of PI3K, AP-1, Cox-2 and Cyclin D1 in blank and experimental group were detected by RT-q PCR. The protein expression levels of PI3K, AP-1, Cox-2 and Cyclin D1 in the blank and each experimental group were detected by Western blot. Results 1. The results of MTT assay showed that Lutein could inhibit the proliferation of human breast cancer MCF-7 cells, and the inhibitory effect was time and dose-dependent, that is, the longer the action time and the greater the concentration of the drug, the stronger the inhibitory effect on MCF-7 cells. The results of flow cytometry showed that the levels of ROS in each group were different, and the level of ROS decreased more significantly with the increase of the concentration of lutein. The results of RT-qPCR showed that the level of mRNA expression of Nrf2 and HO-1 in each concentration group was up-regulated as compared with the blank group. Compared with the blank group, the protein expression of HO-1 was up-regulated, the protein content of Nrf2 in the cytoplasm of the cell was basically no difference, and the content of Nrf2 protein was significantly increased by 5. Annexin V-FITC/ PI cell apoptosis test result showed that the 3-MA group of 24h, The apoptosis rate of the group (40. mu.g/ ml) and 3-MA + Lutein (40. mu.g/ ml) was significantly different from that of the blank group (P0.05). The apoptosis rate of the 3-MA + lutein (40. mu.g/ ml) group was significantly higher than that of the lutein (40. mu.g/ ml) group and the 3-MA group (P0.05). In contrast, the group of lutein (40. mu.g/ ml) was not significantly different from that of the 3-MA group. The results of cell cycle test showed that the cells in the G0/ G1 phase of each experimental group were significantly increased compared with the blank group, and the S-phase cells were significantly reduced, and the 3-MA + Lutein group was more significant, and the results showed that PI3K, AP-1, Cox-2, The expression level of m-RNA of Cyclin D1 was lower than that of blank group, and the difference between the group of lutein (40. mu.g/ ml) and 3-MA group was less, and the decrease of 3-MA + Lutein (40. mu.g/ ml) was more significant (P <0.05). The results of Western blot showed that PI3K, AP-1 and Cox-2, The level of the protein expression of Cyclin D1 was lower than that in the blank group; the difference between the group of lutein (40. mu.g/ ml) and the 3-MA group was small, and the decrease of 3-MA + Lutein (40. mu.g/ ml) was more significant (P0.05). Conclusion 1. Lutein can obviously inhibit the proliferation of human breast cancer MCF-7 cells, and the inhibition effect is time and dose-dependent. Lutein can induce Nrf2 expression and nuclear translocation, promote the expression of anti-oxidation enzyme HO-1, and further reduce the level of oxidative stress. The inhibitory effect of lutein on human breast cancer MCF-7 cells may be mediated by up-regulating the expression of Nrf2 in human breast cancer cells, thereby promoting the expression of HO-1, reducing the ROS content in the cell, inhibiting the expression of PI3K, and further leading to down-regulation of the expression of the AP-1 and its downstream target gene.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9

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