粘细菌Sorangium cellulosum AHB103-1产生新型抗肿瘤物质的研究
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摘要
粘细菌是一类非常独特的革兰氏阴性菌,具有复杂的群体行为和产生丰富的药理活性次级代谢物的能力。本论文对Sorangium cellulosum AHB103-1的生物学特性进行了研究,并对它进行了分类鉴定;研究了发酵过程中产生的挥发性物质与抗肿瘤次级代谢物的关系;利用MTT实验法对抗肿瘤次级代谢物进行了活性筛选和分离纯化,得到两种具有较强抗癌活性的物质迪本霉素和米赫霉素;对迪本霉素进行了摇瓶发酵工艺条件的优化;并初步研究了它的抗肿瘤机制。
对Sorangium cellulosum AHB103-1菌株进行形态学和生理生化特征的研究,根据《伯杰细菌鉴定手册》第二版(第二卷,The Proteobacteria,C部分,George M. Garrity,2005),鉴定为纤维堆囊菌(Sorangium sp.)。采用SPME/GC/MS方法对发酵过程中菌体产生的挥发性物质进行了研究,结果表明部分化合物含量的变化曲线与抗肿瘤活性的变化曲线类似,其中乙酸和4-苯甲酰-2氢-吡喃-3-酮最为重要,它们含量的变化可以做为发酵控制的一个指标。
Sorangium cellulosum AHB103-1菌株产生的次级代谢物对人肝癌细胞HepG2、小鼠黑色素瘤B16、人乳腺癌细胞MDA-MB231、人胃癌细胞SGC7901都有很高的活性,次级代谢物在不高于60℃的条件下活性基本没有变化,对强日光和紫外光不敏感,在pH3~8时较稳定。
利用MTT法,以人肝癌细胞HepG2细胞株为模型,采用液液萃取、Sephadex LH-20柱分离、低压C18柱分离和HPLC、结晶等手段制备出2种具有较高抗肿瘤活性的物质,通过对其NMR、TOF MS/MS、UV、IR等图谱的解析,确定了它们的分子量分别为392、370 Da,分子式分别为C24H44N2O2和C18H30N2O6。根据系统命名法和国际惯例,化合物C24H44N2O2的系统名称为3,12-二乙基-1,15-二氮杂双环[13.7.0]二十二烷-2,16-二酮([3S,12R]3,12-diethyl-1,15-diaza-bicyclo [13.7.0]docosane-2,16-dione),拟命名为Dieobone(迪本霉素);化合物C18H30N2O6的系统名称为2,6,7,8,20-五羟基-4,13-二氮杂四环[9.8.1. 04,9.013,19]二十烷-5-酮( [2R,6R,7S,8R,20R]-2,6,7,8,20-pentahydroxy-4,13-diaza -tetracyclic[9.8. 1.04,9.013,19]eicosane-5-one),拟命名为Mehyazaone(米赫霉素)。通过江南大学教育部科技查新文献检索中心对它们进行了查新(名称、结构、性质和研究现状等),查新号为201136000L080497,国内外均未见公开文献报道与之相同结构的物质。
设计了一种以葡萄糖为唯一碳源,以天冬酰胺为唯一氮源,添加1 mg/L CoCl2的简单合成培养基来研究各种营养成分包括乙酸钠、丙酸钠、甘油、各种氨基酸、无机盐以及磷酸盐的影响。简单合成培养基中添加丙酸钠能显著提高迪本霉素含量55.63%,加入乙酸钠只有略微的促进,加入甘油则会强烈抑制迪本霉素产量至46.71%。加入丝氨酸和色氨酸相应提高迪本霉素的产量100.42%和126.90%,加入苏氨酸则降低了迪本霉素产量至47.64%。不同浓度的磷酸盐对迪本霉素的产量都表现出抑制作用。
在复杂发酵培养基(培养基A)发酵过程中,添加效果较好的树脂有四种:HPD100,XAD-2,XAD-16,D101。其中XAD-16大孔树脂效果最好,达54.66 mg/L。XAD-16大孔树脂较佳添加量为15~25 g/L,摇瓶装液量在75~125 mL/500mL时迪本霉素含量较高,发酵温度在28~32℃较佳。
PB实验的结果表明复杂发酵培养基中各因素影响显著的顺序为马铃薯淀粉>MgSO4·7H2O>脱脂奶粉>丝氨酸>丙酸钠>酵母粉。BB实验优化发酵培养基得到迪本霉素产量预测值最大时的各组分浓度:马铃薯淀粉12.49 g/L,脱脂奶粉9.77 g/L,MgSO4·7H2O 1.43 g/L,预测值为99.89 mg/L。验证实验迪本霉素含量为90.42 mg/L,而优化前为55.46 mg/L,提高了63.04%。
迪本霉素和米赫霉素对四种肿瘤细胞株HepG2,MDA-MB231,B16和SGC7901均有良好的抗肿瘤效果。与临床药相比,迪本霉素和米赫霉素对HepG2的细胞毒作用虽然比埃博霉素B低,与表柔比星和紫杉醇类似,但都远高于依立替康和奥沙利铂。而且它们对老鼠正常脾细胞的毒活性远低于HepG2,B16,SGC7901细胞。这表明迪本霉素和米赫霉素在一定的浓度范围内对肿瘤细胞具有选择性的抑制作用,是相对安全的。
倒置显微镜下观察经迪本霉素处理24 h后的HepG2细胞,可发现样品对细胞的生长表现出明显的抑制作用。荧光显微镜下观察发现HepG2细胞经迪本霉素处理后,细胞核变小、皱缩,可见致密强荧光;部分细胞核碎裂,形成凋亡小体,出现小的荧光点。扫描电镜观察发现,对照组HepG2细胞表面布满丝状微绒毛,而经迪本霉素处理后HepG2细胞膜表面微绒毛明显变稀疏直至完全消失,细胞膜上出现较多的凋亡小体,细胞核有碎裂现象出现。琼脂糖凝胶电泳结果显示经迪本霉素处理后HepG2细胞出现明显的DNA条带,包含180~200 bp的寡核苷酸片段。利用流式细胞仪进行检测,结果表明:在试验浓度范围内,迪本霉素通过将细胞周期阻滞在S期来抑制HepG2细胞增殖;通过引起HepG2细胞线粒体膜电位ΔΨm下降但不影响细胞膜完整性诱导凋亡;迪本霉素通过上调Caspase-3蛋白、Bax蛋白和p53蛋白的表达量,下调Bcl-2蛋白的表达量来诱导细胞凋亡。
Myxobacteria are a class of very unique gram-negative bacteria, with complex social behavior and the ability of producing abundant secondary metabolites with pharmacological activity. In this thesis, the biological characteristics of Sorangium cellulosum AHB103-1 were studied and the classification was identified. The relationship between the volatile substances and the anti-tumor secondary metabolites produced during fermentation process was studied. Anti-tumor secondary metabolites were screened by MTT test and two strong anti-tumor substances were purified and named as Dieobone and Mehyazaone. The fermentation process conditions of Dieobone were optimized. Its anti-tumor mechanism was also preliminarily discussed.
AHB103-1 was identified as Sorangium cellulosum(Sorangium sp.) on the basis of morphological, physiological and biochemical characteristics analyse,according to Bergey’s manual of systematic bacteriology (Second Edition,Part C,2005). The volatile substances produced during fermentation period were identified by SPME/GC/MS and the tendency of some volatile compounds was found to be similar to the formation curves of the anti-tumor secondary metabolites. In which acetic acid and 4-(benzoyl)-2H-pyran-3-ketone were the most important because of higher content, which could be used as a parameter of fermentation control.
Secondary metabolites produced by Sorangium cellulosum AHB103-1 have strong antitumor activity against human liver cancer cell line (HepG2), human breast cancer cell line (MDA-MB231) , mouse melanoma cell line (B16) and human gastric cancer cells (SGC7901). The activity of secondary metabolites was changed a little below 60℃. It was not sensitive to strong sunlight and UV light, and remained stable at pH3~8.
HepG2 cells were chosen as a model cell line using MTT method. Two high anti-tumor activity component was obtained by using extraction, column separation by atmospheric Sephadex LH-20 and crude C18, HPLC, and crystallization purification. The data of NMR, TOF MS/MS, UV, and IR spectrums were analysed. The results showed that the molecular weight of two compounds were 392 and 370 Da and their molecular formulae were C24H44N2O2 and C18H30N2O6, respectively. According to systematic nomenclature, the compound C24H44N2O2 was named as [3S,12R]3,12-diethyl-1,15-diaza-bicyclo [13.7.0]docosane-2,16-dione) systematically and supose the common name as Dieobone, and the compound C18H30N2O6 named as [2R,6R,7S,8R,20R]-2,6,7,8,20-pentahydroxy-4,13-diaza -tetracyclic [9.8. 1.04,9.013,19]eicosane-5-one systematically and supose the common name as Mehyazaone. They were identified as a new substance by Station of Science and Technology of Education of Minister, Jiangnan University (Novelty Search No. 201136000L080497).
A simple synthetic medium with glucose as the sole carbon source, asparagine as the sole nitrogen source, and Co ion as main trace elements was developed to study the effects of various nutrients including sodium acetate, propionate, glycerol, the amino acids, inorganic salts and phosphate. Dieobone yield was significantly increased by 55.63% with the addition of sodium propionate,and only a slight promotion with the addition of sodium acetate.However, Dieobone yield was strongly inhibited by 46.71% with the addition of glycerol.Moreover, Dieobone yield was correspondingly increased by 100.42% and 126.90% with the supplement of serine and tryptophan, but reduced by 47.64% with the supplement of threonine.Different concentrations of phosphate showed inhibition effects,too.
Four resins showed good effect for Dieobone yield during fermentation process, namely, HPD100, XAD-2, XAD-16, D101. Macroporous resin XAD-16 showed the best effect with Dieobone level up to 54.66 mg/L. Dieobone yield reached better level with conditions as follows: macroporous resin XAD-16 addition level 15~25 g/L, liquid volume in flask 75~125 mL/500mL and the fermentation temperature 28~32℃.
The results of PB experiment showed that the significantly different order of the factors in the fermentation medium was: potato starch> MgSO4?7H2O> skim milk powder> serine> sodium propionate> yeast. Fermentation medium was optimized by BB experiment. At the optimum concentrations of each component (potato starch 12.49 g/L, skim milk powder 9.77 g/L, MgSO4.7H2O 1.43 g/L), the predictive value of Dieobone was 99.89 mg/L. The yield of Dieobone for validation experiment was 90.42 mg/L, and was increased by 63.04%, comparing to 55.46 mg/L before optimization.
MTT assay result indicated that Dieobone and Mehyazaone had good antitumor effect on four tumor cell lines, namely, HepG2, MDA-MB231, B16 and SGC7901 in vitro. Compared with clinical drugs, the cytotoxic activity of Dieobone and Mehyazaone on HepG2 were lower than Epothilone B, similar with Taxol and Epirubicin, but far higher than Irinotecan and Oxaliplatin. Moreover, their cytotoxic activity on normal mouse spleen cell was far lower than HepG2 cell and B16 cell.
After treatment with Dieobone for 24h HepG2 cells showed significant cell growth inhibition on inverted microscope observation. The nucleus of HepG2 cells treated with Dieobone become smaller, shrunken observed under fluorescence microscope. Strong compact fluorescent was shown and part of the cell nuclei appeared fragmented. The apoptotic bodies formed, and small fluorescent spots appeared. The observation by scanning electron microscopy showed that the treated HepG2 cell surface microvilli became thin or complete disappeared while the control HepG2 cell covered with silk microvilli. More apoptotic bodies occured on the cell membrane, and the fragmentation of nuclei appeared. The results of agarose gel electrophoresis showed the apparent DNA ladder, containing 180~200bp oligonucleotide fragment. The results of flow cytometry analysis showed that Dieobone suppressed HepG2 cell lines proliferation with cell cycle arrested in the S-phase in the experimental concentration range. It induced apoptosis in the experimental concentration range by causing mitochondrial membrane potentialΔΨm to decline, and markedly upregulated the expression of caspase-3, Bax and p53 mRNA, while downregulated the expression of Bcl-2 mRNA.
对Sorangium cellulosum AHB103-1菌株进行形态学和生理生化特征的研究,根据《伯杰细菌鉴定手册》第二版(第二卷,The Proteobacteria,C部分,George M. Garrity,2005),鉴定为纤维堆囊菌(Sorangium sp.)。采用SPME/GC/MS方法对发酵过程中菌体产生的挥发性物质进行了研究,结果表明部分化合物含量的变化曲线与抗肿瘤活性的变化曲线类似,其中乙酸和4-苯甲酰-2氢-吡喃-3-酮最为重要,它们含量的变化可以做为发酵控制的一个指标。
Sorangium cellulosum AHB103-1菌株产生的次级代谢物对人肝癌细胞HepG2、小鼠黑色素瘤B16、人乳腺癌细胞MDA-MB231、人胃癌细胞SGC7901都有很高的活性,次级代谢物在不高于60℃的条件下活性基本没有变化,对强日光和紫外光不敏感,在pH3~8时较稳定。
利用MTT法,以人肝癌细胞HepG2细胞株为模型,采用液液萃取、Sephadex LH-20柱分离、低压C18柱分离和HPLC、结晶等手段制备出2种具有较高抗肿瘤活性的物质,通过对其NMR、TOF MS/MS、UV、IR等图谱的解析,确定了它们的分子量分别为392、370 Da,分子式分别为C24H44N2O2和C18H30N2O6。根据系统命名法和国际惯例,化合物C24H44N2O2的系统名称为3,12-二乙基-1,15-二氮杂双环[13.7.0]二十二烷-2,16-二酮([3S,12R]3,12-diethyl-1,15-diaza-bicyclo [13.7.0]docosane-2,16-dione),拟命名为Dieobone(迪本霉素);化合物C18H30N2O6的系统名称为2,6,7,8,20-五羟基-4,13-二氮杂四环[9.8.1. 04,9.013,19]二十烷-5-酮( [2R,6R,7S,8R,20R]-2,6,7,8,20-pentahydroxy-4,13-diaza -tetracyclic[9.8. 1.04,9.013,19]eicosane-5-one),拟命名为Mehyazaone(米赫霉素)。通过江南大学教育部科技查新文献检索中心对它们进行了查新(名称、结构、性质和研究现状等),查新号为201136000L080497,国内外均未见公开文献报道与之相同结构的物质。
设计了一种以葡萄糖为唯一碳源,以天冬酰胺为唯一氮源,添加1 mg/L CoCl2的简单合成培养基来研究各种营养成分包括乙酸钠、丙酸钠、甘油、各种氨基酸、无机盐以及磷酸盐的影响。简单合成培养基中添加丙酸钠能显著提高迪本霉素含量55.63%,加入乙酸钠只有略微的促进,加入甘油则会强烈抑制迪本霉素产量至46.71%。加入丝氨酸和色氨酸相应提高迪本霉素的产量100.42%和126.90%,加入苏氨酸则降低了迪本霉素产量至47.64%。不同浓度的磷酸盐对迪本霉素的产量都表现出抑制作用。
在复杂发酵培养基(培养基A)发酵过程中,添加效果较好的树脂有四种:HPD100,XAD-2,XAD-16,D101。其中XAD-16大孔树脂效果最好,达54.66 mg/L。XAD-16大孔树脂较佳添加量为15~25 g/L,摇瓶装液量在75~125 mL/500mL时迪本霉素含量较高,发酵温度在28~32℃较佳。
PB实验的结果表明复杂发酵培养基中各因素影响显著的顺序为马铃薯淀粉>MgSO4·7H2O>脱脂奶粉>丝氨酸>丙酸钠>酵母粉。BB实验优化发酵培养基得到迪本霉素产量预测值最大时的各组分浓度:马铃薯淀粉12.49 g/L,脱脂奶粉9.77 g/L,MgSO4·7H2O 1.43 g/L,预测值为99.89 mg/L。验证实验迪本霉素含量为90.42 mg/L,而优化前为55.46 mg/L,提高了63.04%。
迪本霉素和米赫霉素对四种肿瘤细胞株HepG2,MDA-MB231,B16和SGC7901均有良好的抗肿瘤效果。与临床药相比,迪本霉素和米赫霉素对HepG2的细胞毒作用虽然比埃博霉素B低,与表柔比星和紫杉醇类似,但都远高于依立替康和奥沙利铂。而且它们对老鼠正常脾细胞的毒活性远低于HepG2,B16,SGC7901细胞。这表明迪本霉素和米赫霉素在一定的浓度范围内对肿瘤细胞具有选择性的抑制作用,是相对安全的。
倒置显微镜下观察经迪本霉素处理24 h后的HepG2细胞,可发现样品对细胞的生长表现出明显的抑制作用。荧光显微镜下观察发现HepG2细胞经迪本霉素处理后,细胞核变小、皱缩,可见致密强荧光;部分细胞核碎裂,形成凋亡小体,出现小的荧光点。扫描电镜观察发现,对照组HepG2细胞表面布满丝状微绒毛,而经迪本霉素处理后HepG2细胞膜表面微绒毛明显变稀疏直至完全消失,细胞膜上出现较多的凋亡小体,细胞核有碎裂现象出现。琼脂糖凝胶电泳结果显示经迪本霉素处理后HepG2细胞出现明显的DNA条带,包含180~200 bp的寡核苷酸片段。利用流式细胞仪进行检测,结果表明:在试验浓度范围内,迪本霉素通过将细胞周期阻滞在S期来抑制HepG2细胞增殖;通过引起HepG2细胞线粒体膜电位ΔΨm下降但不影响细胞膜完整性诱导凋亡;迪本霉素通过上调Caspase-3蛋白、Bax蛋白和p53蛋白的表达量,下调Bcl-2蛋白的表达量来诱导细胞凋亡。
Myxobacteria are a class of very unique gram-negative bacteria, with complex social behavior and the ability of producing abundant secondary metabolites with pharmacological activity. In this thesis, the biological characteristics of Sorangium cellulosum AHB103-1 were studied and the classification was identified. The relationship between the volatile substances and the anti-tumor secondary metabolites produced during fermentation process was studied. Anti-tumor secondary metabolites were screened by MTT test and two strong anti-tumor substances were purified and named as Dieobone and Mehyazaone. The fermentation process conditions of Dieobone were optimized. Its anti-tumor mechanism was also preliminarily discussed.
AHB103-1 was identified as Sorangium cellulosum(Sorangium sp.) on the basis of morphological, physiological and biochemical characteristics analyse,according to Bergey’s manual of systematic bacteriology (Second Edition,Part C,2005). The volatile substances produced during fermentation period were identified by SPME/GC/MS and the tendency of some volatile compounds was found to be similar to the formation curves of the anti-tumor secondary metabolites. In which acetic acid and 4-(benzoyl)-2H-pyran-3-ketone were the most important because of higher content, which could be used as a parameter of fermentation control.
Secondary metabolites produced by Sorangium cellulosum AHB103-1 have strong antitumor activity against human liver cancer cell line (HepG2), human breast cancer cell line (MDA-MB231) , mouse melanoma cell line (B16) and human gastric cancer cells (SGC7901). The activity of secondary metabolites was changed a little below 60℃. It was not sensitive to strong sunlight and UV light, and remained stable at pH3~8.
HepG2 cells were chosen as a model cell line using MTT method. Two high anti-tumor activity component was obtained by using extraction, column separation by atmospheric Sephadex LH-20 and crude C18, HPLC, and crystallization purification. The data of NMR, TOF MS/MS, UV, and IR spectrums were analysed. The results showed that the molecular weight of two compounds were 392 and 370 Da and their molecular formulae were C24H44N2O2 and C18H30N2O6, respectively. According to systematic nomenclature, the compound C24H44N2O2 was named as [3S,12R]3,12-diethyl-1,15-diaza-bicyclo [13.7.0]docosane-2,16-dione) systematically and supose the common name as Dieobone, and the compound C18H30N2O6 named as [2R,6R,7S,8R,20R]-2,6,7,8,20-pentahydroxy-4,13-diaza -tetracyclic [9.8. 1.04,9.013,19]eicosane-5-one systematically and supose the common name as Mehyazaone. They were identified as a new substance by Station of Science and Technology of Education of Minister, Jiangnan University (Novelty Search No. 201136000L080497).
A simple synthetic medium with glucose as the sole carbon source, asparagine as the sole nitrogen source, and Co ion as main trace elements was developed to study the effects of various nutrients including sodium acetate, propionate, glycerol, the amino acids, inorganic salts and phosphate. Dieobone yield was significantly increased by 55.63% with the addition of sodium propionate,and only a slight promotion with the addition of sodium acetate.However, Dieobone yield was strongly inhibited by 46.71% with the addition of glycerol.Moreover, Dieobone yield was correspondingly increased by 100.42% and 126.90% with the supplement of serine and tryptophan, but reduced by 47.64% with the supplement of threonine.Different concentrations of phosphate showed inhibition effects,too.
Four resins showed good effect for Dieobone yield during fermentation process, namely, HPD100, XAD-2, XAD-16, D101. Macroporous resin XAD-16 showed the best effect with Dieobone level up to 54.66 mg/L. Dieobone yield reached better level with conditions as follows: macroporous resin XAD-16 addition level 15~25 g/L, liquid volume in flask 75~125 mL/500mL and the fermentation temperature 28~32℃.
The results of PB experiment showed that the significantly different order of the factors in the fermentation medium was: potato starch> MgSO4?7H2O> skim milk powder> serine> sodium propionate> yeast. Fermentation medium was optimized by BB experiment. At the optimum concentrations of each component (potato starch 12.49 g/L, skim milk powder 9.77 g/L, MgSO4.7H2O 1.43 g/L), the predictive value of Dieobone was 99.89 mg/L. The yield of Dieobone for validation experiment was 90.42 mg/L, and was increased by 63.04%, comparing to 55.46 mg/L before optimization.
MTT assay result indicated that Dieobone and Mehyazaone had good antitumor effect on four tumor cell lines, namely, HepG2, MDA-MB231, B16 and SGC7901 in vitro. Compared with clinical drugs, the cytotoxic activity of Dieobone and Mehyazaone on HepG2 were lower than Epothilone B, similar with Taxol and Epirubicin, but far higher than Irinotecan and Oxaliplatin. Moreover, their cytotoxic activity on normal mouse spleen cell was far lower than HepG2 cell and B16 cell.
After treatment with Dieobone for 24h HepG2 cells showed significant cell growth inhibition on inverted microscope observation. The nucleus of HepG2 cells treated with Dieobone become smaller, shrunken observed under fluorescence microscope. Strong compact fluorescent was shown and part of the cell nuclei appeared fragmented. The apoptotic bodies formed, and small fluorescent spots appeared. The observation by scanning electron microscopy showed that the treated HepG2 cell surface microvilli became thin or complete disappeared while the control HepG2 cell covered with silk microvilli. More apoptotic bodies occured on the cell membrane, and the fragmentation of nuclei appeared. The results of agarose gel electrophoresis showed the apparent DNA ladder, containing 180~200bp oligonucleotide fragment. The results of flow cytometry analysis showed that Dieobone suppressed HepG2 cell lines proliferation with cell cycle arrested in the S-phase in the experimental concentration range. It induced apoptosis in the experimental concentration range by causing mitochondrial membrane potentialΔΨm to decline, and markedly upregulated the expression of caspase-3, Bax and p53 mRNA, while downregulated the expression of Bcl-2 mRNA.
引文
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