本文選題：三峽庫區(qū)土壤 + Aspergillus��； 參考：《西南大學(xué)》2011年碩士論文

【摘要】：研究背景：近年來,隨著各種抗生素的廣泛應(yīng)用,病原微生物的耐藥問題日趨嚴重,尋找新型有效抗生素刻不容緩；抗腫瘤藥物雖種類繁多,但其毒副作用、高昂成本也迫使人們?nèi)ふ倚滦陀行Э鼓[瘤藥物,因此國內(nèi)外科研工作者正從天然產(chǎn)物中積極探索、尋找活性先導(dǎo)化合物。真菌特有的遺傳背景和生物合成途徑,使其次級代謝產(chǎn)物極為豐富,成為篩選新型抗菌藥物的和抗腫瘤活性先導(dǎo)化合物的重要來源。自第一個廣譜抗生素—青霉素問世以來,從真菌中尋找具有生物活性、結(jié)構(gòu)新穎的次級代謝產(chǎn)物的研究長期受到生物醫(yī)藥工作者的重視。 三峽庫區(qū)地處四川盆地與長江中下游平原的交界處,巫山地處三峽庫區(qū)腹心,屬于亞熱帶季風濕潤氣候,雨量充沛,獨特的喀斯特地貌和明顯的立體氣候特征使巫山地區(qū)土壤蘊藏著豐富的微生物資源,各種微生物可能因環(huán)境的特殊而代謝多種活性次級代謝產(chǎn)物,成為新藥篩選中重要的先導(dǎo)化合物來源。 研究目的：本文從三峽庫區(qū)巫山土壤樣品中分離培養(yǎng)真菌,經(jīng)過抗菌活性、抗癌活性篩選后,選擇活性抗菌譜廣、活性較強的菌株作為研究對象,對其次級代謝產(chǎn)物進行初步研究,以獲得活性產(chǎn)物,尋找先導(dǎo)化合物,豐富三峽庫區(qū)天然藥用先導(dǎo)化合物的來源。 研究方法：本文對采集的土壤樣品進行處理后,利用平板稀釋法進行真菌的分離培養(yǎng)；對所得到的真菌菌株采用牛津杯雙層平板法和MTT法,以金黃色葡萄球菌(Staphylococcus aureus)、大腸桿菌(Escherichia coli)、白色念珠菌(Candida albicans)、藤黃八疊球菌(Sarcina lutea)、枯草芽孢桿菌(Bacillus subtilis)為受試細菌,結(jié)腸癌細胞SW480為受試細胞,進行抗菌活性和抗癌活性篩選；對菌株SXW-05在三種鑒定培養(yǎng)基(PDA培養(yǎng)基、查氏培養(yǎng)基、馬丁培養(yǎng)基)上的菌落培養(yǎng)特征、顯微形態(tài)特征的觀察及18S rDNA序列測定和系統(tǒng)發(fā)育分析,結(jié)合真菌鑒定手冊(魏景超)綜合判斷,確定該菌株的種屬地位；以PDA液體培養(yǎng)基中25℃,210r/min發(fā)酵8天后所得產(chǎn)物用乙酸乙酯充分萃取,濃縮成浸膏,采用用薄層層析、柱層析、高效液相色譜、重結(jié)晶等分離方法結(jié)合活性追蹤對其活性產(chǎn)物進行分離純化；利用質(zhì)譜、紫外、核磁共振等波譜手段對所得活性單體進行結(jié)構(gòu)鑒定；采用MTT法對單體進行抗腫瘤活性評價,以宮頸癌Hela、結(jié)腸癌細胞SW480為受試細胞,測定該化合物對這兩種癌細胞的增殖抑制率,對活性單體抗腫瘤活性的實現(xiàn)初步評價。 研究結(jié)果：本文從三峽庫區(qū)巫山土壤樣品中共分離獲得形態(tài)各異的真菌20株。經(jīng)活性篩選后發(fā)現(xiàn)一株發(fā)酵產(chǎn)物對金黃色葡萄球菌(Staphylococcus aureus)、大腸桿菌(Escherichia coli)、白色念珠菌(Candida albicans)、藤黃八疊球菌(Sarcina lutea)均有明顯抑制作用,并且對結(jié)腸癌細胞SW480表現(xiàn)出抑制活性的菌株,將其作為為最終研究的目標菌株,并命名為SXW-05。經(jīng)過菌落形態(tài)、顯微形態(tài)觀察,菌種鑒定后,確定該菌株與土曲霉Aspergillus terreus同源性最高,為98%,并且與Aspergillus terreus FJ878634.1處于同一分支,親緣關(guān)系最近。對菌株SXW-05進行菌落觀察時,發(fā)現(xiàn)在PDA固體平板上生長情況最為良好,因此最終確定該菌侏的發(fā)酵條件為：PDA液體培養(yǎng)基中25℃,210r/min發(fā)酵8天,經(jīng)過乙酸乙酯充分萃取后得到浸膏5kg,將其分離純化后最終得到一個有抗腫瘤活性單體FB44,對其結(jié)構(gòu)鑒定后確定該化合物為4,5-二羥基-3-丙烯基-1-環(huán)戊烯基-1-酮,與文獻報道的terrein結(jié)構(gòu)一致。對單體FB44進行抗腫瘤活性評價,以宮頸癌Hela、結(jié)腸癌細胞SW480、肺癌細胞A549為受試細胞,測定化合物FB44分別對兩種癌細胞的增值抑制率,測定其IC5o值分別為0.03mM、0.23mM、9μM,表現(xiàn)出良好的抗腫瘤活性。 結(jié)論：本文最終從三峽庫區(qū)土壤分離到具有抗腫瘤活性的真菌Aspergillus terreusSXW-05,并得到其主要代謝產(chǎn)物之一化合物4,5-二羥基-3-丙烯基-2-環(huán)戊烯基-1-酮,為后續(xù)研究該化合物其他生理活性以及活性機理奠定了基礎(chǔ),豐富了三峽庫區(qū)天然藥用先導(dǎo)化合物的來源。
[Abstract]:Research background: in recent years, with the wide application of various antibiotics, the drug resistance of pathogenic microbes is becoming more and more serious. It is urgent to find new effective antibiotics. Although there are many kinds of antitumor drugs, their toxic side effects and high cost also force people to find new effective antitumor drugs. Therefore, researchers at home and abroad are in the right place. The natural products are actively exploring and searching for active precursor compounds. The specific genetic background and biosynthesis pathway of fungi are very rich in secondary metabolites and become an important source of screening new antiseptic drugs and antitumor precursor compounds. Since the first broad-spectrum antibiotic - penicillin came out, it has been found from fungi. Biopharmaceutical workers have attached great importance to the research of secondary metabolites with biological activity and novel structure.
The Three Gorges Reservoir area is located at the junction of the Sichuan basin and the plain of the middle and lower reaches of the Yangtze River. Wushan is located in the heart of the Three Gorges Reservoir area. It belongs to the subtropical monsoon humid climate. The rainfall is abundant, the unique Karst landforms and obvious three-dimensional climatic characteristics make the soil of Wushan rich in microbial resources. All kinds of microorganisms may be replaced by the special environment. Thanks to many active secondary metabolites, it has become an important lead compound source in new drug screening.
The purpose of this study is to isolate and culture fungi from the soil samples of Wushan in the Three Gorges Reservoir area. After screening the antibacterial activity and anti-cancer activity, this paper selects the active antibacterial strain as the research object and studies its secondary metabolites, in order to obtain the active products, find the precursor compounds and enrich the natural medicine of the Three Gorges Reservoir area. The source of the pilot compound.
Research methods: after processing the collected soil samples, the fungi were separated and cultivated by plate dilution method. The obtained fungal strains were used in Oxford cup double deck plate method and MTT method, with Staphylococcus aureus (Staphylococcus aureus), Escherichia coli, Candida albicans (Candida albicans), and Garcinia. Sarcina lutea (Sarcina lutea) and Bacillus subtilis (Bacillus subtilis) were tested bacteria, and colon cancer cell SW480 was tested as tested cells to conduct antimicrobial activity and screening of anticancer activity. The micromorphological characteristics of strain SXW-05 in the culture medium (PDA medium, chagfish medium, Martin medium) and microscopic morphological characteristics were observed and observed. 18S rDNA sequence determination and phylogenetic analysis, combined with the comprehensive judgment of the fungal identification manual (Wei Jingchao), determine the species status of the strain; extract the products from PDA liquid medium at 25 and 210r/min after 8 days, and concentrate them into the extract by ethyl acetate, and use thin layer chromatography, column chromatography, high performance liquid chromatography, recrystallization and other separation. Methods the active products were isolated and purified by combined activity tracing. The structure of the active monomers was identified by mass spectrometry, ultraviolet and nuclear magnetic resonance spectroscopy. The anti-tumor activity of the monomers was evaluated by MTT. The Hela of cervical cancer and SW480 of colon cancer cells were tested, and the increase of the compound to these two kinds of cancer cells was determined. Inhibition rate of colonization and preliminary evaluation of the anti-tumor activity of active monomer.
Results: 20 strains of different fungi were isolated from the Wushan soil samples in the Three Gorges Reservoir area. After active screening, a strain of Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Candida albicans (Candida albicans), and eight Staph (Sarcina lutea) were found to be obvious after screening. The inhibitory activity, and the strain that showed inhibitory activity to the colon cancer cell SW480, was used as the target strain for the final study. The strain was named as SXW-05. through colony morphology, microscopic observation, and after identification, it was identified that the strain was the highest homology to Aspergillus Aspergillus terreus, 98%, and Aspergillus terreus FJ878634.1 In the same branch, close relatives recently. When the strain SXW-05 was observed in the colony, it was found that the growth of the PDA solid plate was the best. Therefore, the fermentation conditions of the bacteria were determined as follows: 25 C in the PDA liquid medium, 8 days for the fermentation of the bacteria, and the extract 5kg after the full extraction of ethyl acetate. An anti-tumor active monomer FB44 was obtained. After identification of its structure, the compound was identified as 4,5- two hydroxyl -3- propyl -1- cyclopentenyl -1- ketone, consistent with the terrein structure reported in the literature. The anti-tumor activity of the monomer FB44 was evaluated, the cervical cancer Hela, the colon cancer cell SW480, and the lung cancer cell A549 were tested, and the compound FB44 was tested for FB44. The inhibition rates of two kinds of cancer cells were measured respectively, and their IC5o values were 0.03mM, 0.23mM and 9 M respectively, showing a good antitumor activity.
Conclusion: in this paper, Aspergillus terreusSXW-05 with antitumor activity was isolated from the soil of the Three Gorges Reservoir area, and 4,5- two hydroxyl -3- propene -2- cyclopentenyl -1- ketone, one of its main metabolites, was obtained. It laid the foundation for further study of other physiological activities and active mechanism of the compound, enriching the three Gorges Reservoir area. Sources of natural medicinal precursor compounds.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R379

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本文編號：1895786