铜绿假单胞菌代谢产物抗肿瘤活性研究

发布时间：2018-04-19 13:53

本文选题：铜绿假单胞菌 + 双鼠李糖脂　；参考：《南京理工大学》2014年硕士论文

【摘要】：铜绿假单胞菌(Pseudomonas aeruginosa)是一种常见的革兰氏阴性菌,其次级代谢产物在环境、工业、医药等行业有良好的应用前景。如鼠李糖脂作为一种用途广泛的生物表面活性剂,兼具加快伤口愈合、防治藻类、抑制微生物生长等生物活性；绿脓菌素是一种小分子吩嗪类蓝色色素,具有较强的氧化还原活性,可扩散并渗透进入细胞膜,过量产生氧自由基导致细胞死亡。本研究对模拟空间环境条件诱变得到的高产鼠李糖脂突变菌株P. aeruginosa M14808进行发酵,分离、纯化其次级代谢产物并表征鼠李糖脂的化学结构,研究鼠李糖脂及绿脓菌素的抗肿瘤活性,并探讨其作用机制。主要研究结果如下： 1.结构表征结果表明,从发酵液中分离、纯化得到的鼠李糖脂主要为双鼠李糖脂Rh2C10C10。该物质对人乳腺癌细胞MCF-7增殖的抑制作用明显,可增强衰老相关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基因表达水平的检测结果表明,双鼠李糖脂促使MCF-7细胞坏死。 2.对种子液进行分离、纯化,得到绿脓菌素。绿脓菌素能够抑制人非小细胞肺癌细胞A549及人肝癌细胞HepG2的生长,且呈剂量依赖关系。AO/EB染色、DNA ladder及单细胞凝胶电泳检测提示绿脓菌素可诱导肿瘤细胞发生凋亡。进一步研究表明绿脓菌素能诱导A549和HepG2细胞内ROS水平升高,caspase-3活性增强,抗氧化物质含量随之改变。HepG2细胞内p53、caspase-9、bax及PIG3mRNA表达水平显著升高, bcl-2/bax比值降低。抗氧化剂N-乙酰基-L-半胱氨酸(NAC)预处理后,A549和HepG2细胞的生存能力显著增强,氧化应激相关指标及各基因表达水平均有所改善。实验结果表明：绿脓菌素诱导A549和HepG2细胞产生氧化应激,并进而引起HepG2细胞内p53基因和线粒体途径介导的细胞凋亡。
[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.
【学位授予单位】：南京理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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