紫色杆菌素和脱氧紫色杆菌素对结肠癌HT29细胞抑癌作用和初步机制研究

发布时间：2018-06-26 23:52

本文选题：紫色杆菌素 + 脱氧紫色杆菌素　；参考：《扬州大学》2017年硕士论文

【摘要】：研究背景:结肠癌(colorectal cancer,CRC)是最常见的恶性肿瘤之一,近年来发病率和死亡率总体呈上升趋势,位列全球女性和男性常见恶性肿瘤的第二位和第三位。在我国,随着人们膳食结构和生活方式的改变,结肠癌发病率逐年上升,且趋于年轻化。目前,针对结肠癌的主要治疗手段为手术切除,辅以放化疗及生物治疗等,但治疗效果通常不理想,辅助治疗产生的副作用大,预后较差。紫色杆菌素和脱氧紫色杆菌素作为一种蓝紫色的微生物代谢产物,着色力强,并且兼具抗肿瘤、抗革兰氏阳性菌和抗植物致病菌等活性,属于低基因毒性物质,有希望用作食品着色剂和防腐剂,在医药行业也同样具有广泛的应用前景。目的:检测紫色杆菌素和脱氧紫色杆菌素对结肠癌HT29细胞的抑制效果,探索紫色杆菌素和脱氧紫色杆菌素对结肠癌HT29细胞可能的作用机制,为紫色杆菌素和脱氧紫色杆菌素的未来临床应用提供理论基础。方法:用不同浓度的紫色杆菌素或脱氧紫色杆菌素处理结肠癌HT29细胞,采用四甲基偶氮唑蓝(MTT)比色法,检测紫色杆菌素或脱氧紫色杆菌素对HT29细胞的抑制率;用倒置相差显微镜观察紫色杆菌素或脱氧紫色杆菌素对HT29细胞的形态学改变;流式细胞术检测细胞周期和凋亡;Western Blot法检测凋亡相关蛋白Bax、Bcl-2和Caspase9表达的变化。同时用透射电镜进一步观察细胞的超微结构变化,用Western Blot法检测Beclin1和P62蛋白是否参与紫色杆菌素或脱氧紫色杆菌素诱导的自噬过程。结果:紫色杆菌素和脱氧紫色杆菌素均能显著抑制结肠癌HT29细胞增殖,呈时间剂量依赖性;倒置相差显微镜观察到紫色杆菌素或脱氧紫色杆菌素干预后显著的细胞形态学改变,在细胞质中可见大量的大小不等的空泡,细胞空泡随药物浓度的增加逐渐增大并增多。随着药物浓度的增加,处于G0/G1期的细胞比例明显增加,S期的比例明显减少;紫色杆菌素和脱氧紫色杆菌素均可以增加HT29细胞凋亡比例,使凋亡蛋白Caspase9和Bax表达上升,并下调抑凋亡Bcl-2的表达;透射电镜进一步证实了紫色杆菌素或脱氧紫色杆菌素干预后的HT29细胞内生成了大量的自噬泡,自噬蛋白Beclin1的表达上升。结论:紫色杆菌素和脱氧紫色杆菌素均能够有效地抑制结肠癌HT29细胞增殖并诱导凋亡和自噬,其凋亡机制可能作用于线粒体介导的内源性通路,Beclin1可能也参与了紫色杆菌素或脱氧紫色杆菌素诱导的自噬过程。
[Abstract]:Background: colorectal cancer is one of the most common malignant tumors. In recent years, the morbidity and mortality rate of colorectal cancer is on the rise, and it ranks second and third among the common malignant tumors in women and men. In China, with the change of diet and lifestyle, the incidence of colon cancer is increasing year by year and tends to be younger. At present, the main treatment for colon cancer is surgical resection, combined with radiotherapy and chemotherapy, biological therapy, but the treatment effect is usually not ideal, the side effects of adjuvant treatment are large, the prognosis is poor. As a metabolite of blue-purple microorganism, Purple Bacterin and Deoxypurple Bacteriocin have strong coloring power and have anti-tumor, anti-Gram-positive bacteria and anti-phytopathogenic bacteria activities, so they are low genotoxic substances. It is expected to be used as food colorant and preservative, and also has a wide application prospect in pharmaceutical industry. Objective: to investigate the inhibitory effect of purple bactein and deoxypurple bactein on colon cancer HT29 cells, and to explore the possible mechanism of purple and deoxypurple bacterein on colon cancer HT29 cells. To provide a theoretical basis for the future clinical application of purple bacterein and deoxypurple bactein. Methods: HT29 cells were treated with different concentrations of Purple Bacterin or Deoxypurple Bacterin. MTT colorimetric assay was used to detect the inhibition rate of purple bacterein or deoxypurple bacterein on HT29 cells. The morphological changes of HT29 cells were observed by inverted phase contrast microscope, the cell cycle was detected by flow cytometry and the expression of apoptosis-related proteins Bcl-2 and caspase9 were detected by Western Blot. At the same time, the ultrastructural changes of the cells were observed by transmission electron microscope. Western blot was used to detect whether Beclin1 and P62 proteins were involved in the autophagy induced by Purple Bacterin or deoxypurple Bacterin. Results: both Purple Bacterin and deoxypurple Bacterin could significantly inhibit the proliferation of colon cancer cell line HT29 in a time-dose dependent manner, and the morphological changes were observed by inverted phase contrast microscope. A large number of vacuoles of different sizes were found in the cytoplasm, and the vacuoles increased with the increase of drug concentration. With the increase of drug concentration, the proportion of cells in G _ 0 / G _ 1 phase significantly increased and the proportion of S phase decreased, while both Purple Bacterin and Deoxypurple Bacterin increased the apoptosis rate of HT29 cells, and increased the expression of Caspase9 and Bax, and increased the expression of Caspase9 and Bax in HT29 cells. It was further confirmed by transmission electron microscope that a large number of autophagy was formed in HT29 cells after the intervention of Purple Bacterin or deoxypurple Bacterin, and the expression of autophagy protein Beclin1 was increased. Conclusion: both Purple Bacterin and deoxypurple Bacterin can effectively inhibit the proliferation and induce apoptosis and autophagy of colon cancer HT29 cells. The mechanism of apoptosis may play an important role in mitochondrial mediated endogenous pathway, Beclin1, which may also be involved in the autophagy induced by Purple Bacterin or deoxypurple Bacterin.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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