叶黄素介导AP-1失激活的抗人乳腺癌MCF-7细胞增殖效应及其机制研究

发布时间：2018-11-28 18:57

【摘要】：研究背景:乳腺癌是危害女性健康的常见恶性肿瘤之一,是城市女性最常见的癌症,且发病率和死亡率逐年上升。乳腺癌是一种多因素,多基因相互作用引起的癌症,但病因及发病机制尚未完全清楚。目前乳腺癌的治疗虽已逐渐从简单的手术、化疗转向手术、化疗、放疗以及生物靶向治疗等多种手段综合治疗的方向,但仍会对患者身心造成巨大的创伤。因此,寻找高效、低副作用的天然化学药物对乳腺癌的预防和治疗具有重大意义。叶黄素(Lutein)又称为植物黄体素,是一种具有抗氧化作用的天然植物化合物,大量存在于花卉、蔬果等绿色植物中,尤其在万寿菊中含量最为丰富。人们通过膳食摄取的叶黄素可经由血液输送到各组织。流行病学研究显示,叶黄素在保护视力、预防白内障、延缓动脉粥样硬化和预防肿瘤等多方面具有重要作用。目的:本实验旨在通过检测叶黄素(Lutein)对人乳腺癌MCF-7细胞中激活蛋白1(activator protein 1,AP-1)及其上下游信号通路中相关因子的影响,进而研究叶黄素对人乳腺癌MCF-7细胞增殖的影响及其作用机制,为乳腺癌的化学预防及叶黄素作为低副作用的天然抗癌药物的临床研究提供理论依据。方法:1.MTT法检测不同浓度叶黄素(Lutein)对人乳腺癌MCF-7细胞的增殖抑制率。用不同浓度的叶黄素(0、5、10、20、40、80μg/ml)分别处理MCF-7细胞12、24、48h后,用MTT法检测叶黄素对细胞增殖的抑制率。2.流式细胞术检测不同浓度的叶黄素(0、5、10、20、40、80μg/ml)分别处理MCF-7细胞24h后的活性氧(reactive oxygen species,ROS)水平。3.采用RT-q PCR检测各组细胞中Nrf2、HO-1的m RNA的表达水平。4.采用Western blot检测各组细胞胞浆和细胞核中Nrf2和HO-1的蛋白表达水平。5.根据以上实验结果分析设置空白组、3-MA组、叶黄素组、3-MA+叶黄素组进行后续实验。流式细胞术检测空白组及各实验组细胞周期的变化及凋亡率。6.采用RT-q PCR检测空白组及各实验组细胞中PI3K、AP-1、Cox-2和Cyclin D1的m RNA表达水平。7.采用Western blot检测空白组及各实验组细胞中PI3K、AP-1、Cox-2和Cyclin D1的蛋白表达水平。结果1.MTT检测结果显示,叶黄素能够抑制人乳腺癌MCF-7细胞的增殖,且抑制作用具有时间和剂量依赖性,即作用时间越长,用药浓度越大时,对MCF-7细胞的增殖抑制作用越强。2.流式细胞术检测结果显示,各组细胞中ROS水平有差异,且随着叶黄素浓度的增大,ROS水平降低更显著。3.RT-qPCR的实验结果显示,与空白组相比,各浓度组Nrf2、HO-1的mRNA表达水平均上调。4.Western blot的实验结果显示,与空白组相比,HO-1的蛋白表达量呈上调趋势,细胞胞浆中Nrf2的蛋白含量基本无差异,而核Nrf2蛋白的含量明显上调。5.Annexin V-FITC/PI细胞凋亡检测结果显示,24h的3-MA组、叶黄素组(40μg/ml)和3-MA+叶黄素(40μg/ml)组的细胞凋亡率与空白组相比均有明显差异(P0.05),3-MA+叶黄素(40μg/ml)组细胞凋亡率明显大于叶黄素(40μg/ml)组和3-MA组(P0.05),而叶黄素(40μg/ml)组与3-MA组相比,两组无明显差异。6.细胞周期检测结果显示,与空白组相比,各实验组G0/G1期细胞明显增加,S期细胞明显减少,且3-MA+叶黄素组更显著,表现为显著地G1期阻滞(P㩳0.05)。7.RT-q PCR的实验结果显示,PI3K、AP-1、Cox-2、Cyclin D1的m RNA表达水平各实验组与空白组相比均下调;叶黄素(40μg/ml)组和3-MA组相比差异较小,3-MA+叶黄素(40μg/ml)组下调更显著(P0.05)。8.Western blot的实验结果显示,PI3K、AP-1、Cox-2、Cyclin D1的蛋白表达水平各实验组与空白组相比均下调;叶黄素(40μg/ml)组和3-MA组相比差异较小,3-MA+叶黄素(40μg/ml)组下调相对更显著(P0.05)。结论1.叶黄素可明显抑制人乳腺癌MCF-7细胞的增殖,抑制作用具有时间和剂量依赖性。2.叶黄素可诱导Nrf2的表达和核转位,促进抗氧化酶HO-1的表达,进而降低氧化应激水平。3.叶黄素对人乳腺癌MCF-7细胞增值抑制作用可能是通过上调人乳腺癌细胞内Nrf2的表达,从而促进HO-1的表达,降低细胞内的ROS含量,抑制PI3K的表达,进而导致AP-1及其下游靶基因的的表达下调所介导的。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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