本文選題：銅綠假單胞菌 + 雙鼠李糖脂　； 參考：《南京理工大學(xué)》2014年碩士論文

【摘要】：銅綠假單胞菌(Pseudomonas aeruginosa)是一種常見的革蘭氏陰性菌,其次級代謝產(chǎn)物在環(huán)境、工業(yè)、醫(yī)藥等行業(yè)有良好的應(yīng)用前景。如鼠李糖脂作為一種用途廣泛的生物表面活性劑,兼具加快傷口愈合、防治藻類、抑制微生物生長等生物活性；綠膿菌素是一種小分子吩嗪類藍色色素,具有較強的氧化還原活性,可擴散并滲透進入細胞膜,過量產(chǎn)生氧自由基導(dǎo)致細胞死亡。本研究對模擬空間環(huán)境條件誘變得到的高產(chǎn)鼠李糖脂突變菌株P(guān). aeruginosa M14808進行發(fā)酵,分離、純化其次級代謝產(chǎn)物并表征鼠李糖脂的化學(xué)結(jié)構(gòu),研究鼠李糖脂及綠膿菌素的抗腫瘤活性,并探討其作用機制。主要研究結(jié)果如下： 1.結(jié)構(gòu)表征結(jié)果表明,從發(fā)酵液中分離、純化得到的鼠李糖脂主要為雙鼠李糖脂Rh2C10C10。該物質(zhì)對人乳腺癌細胞MCF-7增殖的抑制作用明顯,可增強衰老相關(guān)β-半乳糖苷酶活性。AO/EB染色、DNA ladder、caspase-3活性及p53、bax、bcl-2、casepase-3、 caspase-9、Fas基因表達水平的檢測結(jié)果表明,雙鼠李糖脂促使MCF-7細胞壞死。 2.對種子液進行分離、純化,得到綠膿菌素。綠膿菌素能夠抑制人非小細胞肺癌細胞A549及人肝癌細胞HepG2的生長,且呈劑量依賴關(guān)系。AO/EB染色、DNA ladder及單細胞凝膠電泳檢測提示綠膿菌素可誘導(dǎo)腫瘤細胞發(fā)生凋亡。進一步研究表明綠膿菌素能誘導(dǎo)A549和HepG2細胞內(nèi)ROS水平升高,caspase-3活性增強,抗氧化物質(zhì)含量隨之改變。HepG2細胞內(nèi)p53、caspase-9、bax及PIG3mRNA表達水平顯著升高, bcl-2/bax比值降低。抗氧化劑N-乙�；�-L-半胱氨酸(NAC)預(yù)處理后,A549和HepG2細胞的生存能力顯著增強,氧化應(yīng)激相關(guān)指標及各基因表達水平均有所改善。實驗結(jié)果表明：綠膿菌素誘導(dǎo)A549和HepG2細胞產(chǎn)生氧化應(yīng)激,并進而引起HepG2細胞內(nèi)p53基因和線粒體途徑介導(dǎo)的細胞凋亡。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a common gram-negative bacteria, and its secondary metabolites have a good prospect in environmental, industrial, pharmaceutical and other industries.For example, rhamnolipid, as a widely used biosurfactant, has biological activities such as accelerating wound healing, preventing algae, inhibiting microbial growth, etc. Pseudomonas aeruginosa is a small molecule of phenazine blue pigment.It has strong redox activity, can diffuse and penetrate into cell membrane, and excessive production of oxygen free radicals leads to cell death.In this study, the mutant strain P. M14808, which was induced by simulated space environment conditions, was fermented, separated, purified, and characterized the chemical structure of rhamnolipid.To study the anti-tumor activity of rhamnolipid and pyocyanin and its mechanism.The main findings are as follows:1.The results of structural characterization showed that the purified rhamnolipid was mainly Rh2C10C10C10 from fermentation broth.The inhibitory effect of the compound on MCF-7 proliferation of human breast cancer cells was obvious, and it could enhance the activity of 尾 -galactosidase. AO-P / EB staining and the expression of p53 Baxbax-bcl-2caspase-3, caspase-9 FAS gene in MCF-7 cells. The results showed that rhamnolipid promoted the necrosis of MCF-7 cells.2.The seed liquid was separated and purified to obtain pyogenin.Pseudomonas aeruginosa can inhibit the growth of human non-small cell lung cancer cell line A549 and human hepatoma cell line HepG2 in a dose-dependent manner. The results of ladder staining and single cell gel electrophoresis indicate that pseudomonas aeruginosa can induce apoptosis of tumor cells.Further studies showed that pyocyanin could induce the increase of ROS level in A549 and HepG2 cells and increase the activity of caspase-3. The content of antioxidants in HepG2 cells increased significantly the expression of p53 caspase-9 PIG3mRNA and the ratio of bcl-2/bax decreased.The survival ability of A549 and HepG2 cells was significantly enhanced after pretreatment with the antioxidant N-acetyl-L-cysteine, and the related indexes of oxidative stress and the expression level of each gene were improved.The results showed that Pseudomonas aeruginosa induced oxidative stress in A549 and HepG2 cells and induced apoptosis mediated by p53 gene and mitochondrial pathway in HepG2 cells.
【學(xué)位授予單位】：南京理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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