本文選題：紫色桿菌素 + 脫氧紫色桿菌素��； 參考：《揚州大學》2017年碩士論文

【摘要】：研究背景:結腸癌(colorectal cancer,CRC)是最常見的惡性腫瘤之一,近年來發(fā)病率和死亡率總體呈上升趨勢,位列全球女性和男性常見惡性腫瘤的第二位和第三位。在我國,隨著人們膳食結構和生活方式的改變,結腸癌發(fā)病率逐年上升,且趨于年輕化。目前,針對結腸癌的主要治療手段為手術切除,輔以放化療及生物治療等,但治療效果通常不理想,輔助治療產生的副作用大,預后較差。紫色桿菌素和脫氧紫色桿菌素作為一種藍紫色的微生物代謝產物,著色力強,并且兼具抗腫瘤、抗革蘭氏陽性菌和抗植物致病菌等活性,屬于低基因毒性物質,有希望用作食品著色劑和防腐劑,在醫(yī)藥行業(yè)也同樣具有廣泛的應用前景。目的:檢測紫色桿菌素和脫氧紫色桿菌素對結腸癌HT29細胞的抑制效果,探索紫色桿菌素和脫氧紫色桿菌素對結腸癌HT29細胞可能的作用機制,為紫色桿菌素和脫氧紫色桿菌素的未來臨床應用提供理論基礎。方法:用不同濃度的紫色桿菌素或脫氧紫色桿菌素處理結腸癌HT29細胞,采用四甲基偶氮唑藍(MTT)比色法,檢測紫色桿菌素或脫氧紫色桿菌素對HT29細胞的抑制率;用倒置相差顯微鏡觀察紫色桿菌素或脫氧紫色桿菌素對HT29細胞的形態(tài)學改變;流式細胞術檢測細胞周期和凋亡;Western Blot法檢測凋亡相關蛋白Bax、Bcl-2和Caspase9表達的變化。同時用透射電鏡進一步觀察細胞的超微結構變化,用Western Blot法檢測Beclin1和P62蛋白是否參與紫色桿菌素或脫氧紫色桿菌素誘導的自噬過程。結果:紫色桿菌素和脫氧紫色桿菌素均能顯著抑制結腸癌HT29細胞增殖,呈時間劑量依賴性;倒置相差顯微鏡觀察到紫色桿菌素或脫氧紫色桿菌素干預后顯著的細胞形態(tài)學改變,在細胞質中可見大量的大小不等的空泡,細胞空泡隨藥物濃度的增加逐漸增大并增多。隨著藥物濃度的增加,處于G0/G1期的細胞比例明顯增加,S期的比例明顯減少;紫色桿菌素和脫氧紫色桿菌素均可以增加HT29細胞凋亡比例,使凋亡蛋白Caspase9和Bax表達上升,并下調抑凋亡Bcl-2的表達;透射電鏡進一步證實了紫色桿菌素或脫氧紫色桿菌素干預后的HT29細胞內生成了大量的自噬泡,自噬蛋白Beclin1的表達上升。結論:紫色桿菌素和脫氧紫色桿菌素均能夠有效地抑制結腸癌HT29細胞增殖并誘導凋亡和自噬,其凋亡機制可能作用于線粒體介導的內源性通路,Beclin1可能也參與了紫色桿菌素或脫氧紫色桿菌素誘導的自噬過程。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-3

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