粘细菌抗肿瘤活性物质筛选及对荷瘤小鼠抑瘤活性和机制研究
摘要
本论文对河北省微生物多样性研究与应用实验室保藏的100株粘细菌的发酵产物进行了体外细胞毒筛选，100个发酵液样品中对人乳腺癌细胞MCF-7有抑制作用的达到82%，说明粘细菌次级代谢产物对肿瘤细胞具有普遍的细胞毒作用。一个好的抗肿瘤药物不仅需要对肿瘤细胞有强大的杀伤作用，而且需对人正常细胞有很小的损害，通过对正常二倍体人胚肺成纤维细胞存活率的测定，选取2~#、15~#、24~#、37~#、66~#、87~#等6株粘细菌的发酵产物，通过利用人子宫颈癌HeLa细胞和人肺腺癌A549进行复筛，结果显示15~#菌株发酵产物对A549和HeLa抑制作用较强，72h的IC50分别为6.66μg/mL和14.70μg/mL，且其抑制作用具有较好的时效关系，对人正常二倍体MRC细胞毒性较小，其实验重复性好，初筛和复筛结果相互印证，有开发成为抗肿瘤药物的潜能，选择进行下一步组分分析。
选择甲醇和乙腈作为流动相分别对15~#菌株发酵代谢产物进行梯度洗脱，通过比较可知乙腈对代谢产物的洗脱能力较强，确定15~#菌株发酵代谢产物的最适分离条件为乙腈：水=49:51（体积比），检测波长为350nm，15~#菌株发酵产物分离得到了5个组分，对人乳腺癌细胞MCF-7的抑制实验结果表明：15-1组分具有很好的量效对应关系，当终浓度为100μg/mL时，15-1对人乳腺癌细胞的抑制率分别为71.73%，其它组分的抑制效果则不明显，说明15-1组分抑制肿瘤细胞生长的活性是较强的。
菌株91018是本实验室已筛选出来的具有抗肿瘤活性的菌株，根据已摸索好的液相分离条件，制备菌株91018发酵产物单组分91018-1，当终浓度为100μg/mL时，对人子宫颈癌细胞和人乳腺癌细胞的抑制率分别为69.19%和22.59%。
荷瘤小鼠抑瘤实验显示91018-1单组分1.25mg/kg剂量组对于小鼠肝癌H_(22)肿瘤细胞的抑瘤率最高，达到48.7%，剂量过大或过小都不利于抑瘤效果。阳性对照环磷酰胺的抑瘤率达到81.3%，但小鼠体征不好。第8天阳性对照组小鼠有一只死亡，说明环磷酰胺的毒性较大，严重影响了小鼠的健康，而1.25mg/kg剂量组小鼠活动自如，皮毛光滑，食量较正常，体重有明显增加。说明1.25mg/kg剂量的91018-1组分对小鼠肝癌H_(22)肿瘤生长有一定的抑制作用，并且对小鼠的毒性较少。流式细胞仪检测发现1.25mg/Kg实验组细胞凋亡率比阴性对照组明显提高，PI细胞增殖指数样品1.25mg/Kg实验组与阴性对照组存在显著差异，说明1.25mg/Kg实验组S和G_2/M期的细胞减少，细胞被阻止在G_0/G_1期，从而影响DNA的合成。1.25mg/Kg样品组与阴性对照组的p53蛋白表达量存在显著差异，说明凋亡通过p53正调控产生，阻止了DNA合成。Bcl-2蛋白表达量与阴性对照相比不存在显著差异，凋亡不是通过Bcl-2蛋白诱导产生。
综合利用质谱(GCT-MS、ESI)、氢谱(~1H-NMR)、碳谱(~(13)C-NMR)、二维谱图(DEPT)等现代波谱技术，对91018-1组分进行结构分析。经解析得知该化合物中文名称为粘噻唑。
In this paper, a total of100strains preserved by the Key Lab of Microbial Diversity Researchand Application of Hebei Province were used to screen anticancer drugs with tumor cell lines byMTT high throughout method.82%strains could produce high anticancer bioactive metaboliteagainst MCF-7tumor cell line. And six strains metabolite had good inhibition effect on MCF-7tumor cell and inhibited human embryo lung cells growth lightly at the same time.
For A549and HeLa cells, strain15had more inhibitive activity. The IC50of72h for A549and HeLa cells were6.66μg/mL and14.70μg/mL respectively. So it was selected for futherinvestigation.
The chromatography method had been studied and the suitable conditions had beendetermined and established. In the method, we used the Thermo ODS-2,5μm,250mm×4.6mmas HPLC column, acetonitrile: water=49:51as mobile phase.5components had been isolatedfrom the fermentation of strain15.
Compound15-1had significant inhibition on breast cancer MCF-7cells. The inhibition ratesof compound15-1on breast cancer MCF-7cells was71.73%, which the concentration was100μg/mL and there was good dose-effect relationship The inhibition rates of the other compoundswere not obvious.
91018-1had been isolated from the fermentation of strain91018which had anticanceractivity. The inhibition rates of compound91018-1on HeLa and MCF-7cells was69.19%and22.59%respectively when the concentration was100μg/mL. Compound91018-1had significantinhibition on breast cancer MCF-7cells.
The H_(22)tumor bearing mice was collected and the anticancer rate was calculated. Resultsshowed the inhibition rate of compound91018-1was48.7%when the dosage was1.25mg/kg.More or less was not better for the inhibition rate. The inhibition rate of positive control whichwas cyclophosphamide was81.3%. But characteristic features of mice were worse. One mousewas dead on the8th day. The1.25mg/kg group had better characteristic features such as behavioractivity, hair and mental status.
The expression of Bcl-2protein and p53protein in the tumor tissue were studied by theflow cytometry method. Results showed91018-1could induce tumor apoptosis by prevention ofcell cycle progression; Cells of S and G_2/M phase decreased. Cells were impeded at G_0/G_1phaseand thus affected DNA synthesis. The apoptosis was conducted by p53protein.
We had utilized modern spectroscopy testing technology such as ESI, GCT-MS,~1H-NMR,~(13)C-NMR and DEPT in order to confirm the structure of91018-1. After analyzing, we knew thatthe molecular weight of91018-1was487.68and the molecular formula was C_(25)H_(33)O_3N_3S_2. Thename of the compound is myxothiazol.
选择甲醇和乙腈作为流动相分别对15~#菌株发酵代谢产物进行梯度洗脱，通过比较可知乙腈对代谢产物的洗脱能力较强，确定15~#菌株发酵代谢产物的最适分离条件为乙腈：水=49:51（体积比），检测波长为350nm，15~#菌株发酵产物分离得到了5个组分，对人乳腺癌细胞MCF-7的抑制实验结果表明：15-1组分具有很好的量效对应关系，当终浓度为100μg/mL时，15-1对人乳腺癌细胞的抑制率分别为71.73%，其它组分的抑制效果则不明显，说明15-1组分抑制肿瘤细胞生长的活性是较强的。
菌株91018是本实验室已筛选出来的具有抗肿瘤活性的菌株，根据已摸索好的液相分离条件，制备菌株91018发酵产物单组分91018-1，当终浓度为100μg/mL时，对人子宫颈癌细胞和人乳腺癌细胞的抑制率分别为69.19%和22.59%。
荷瘤小鼠抑瘤实验显示91018-1单组分1.25mg/kg剂量组对于小鼠肝癌H_(22)肿瘤细胞的抑瘤率最高，达到48.7%，剂量过大或过小都不利于抑瘤效果。阳性对照环磷酰胺的抑瘤率达到81.3%，但小鼠体征不好。第8天阳性对照组小鼠有一只死亡，说明环磷酰胺的毒性较大，严重影响了小鼠的健康，而1.25mg/kg剂量组小鼠活动自如，皮毛光滑，食量较正常，体重有明显增加。说明1.25mg/kg剂量的91018-1组分对小鼠肝癌H_(22)肿瘤生长有一定的抑制作用，并且对小鼠的毒性较少。流式细胞仪检测发现1.25mg/Kg实验组细胞凋亡率比阴性对照组明显提高，PI细胞增殖指数样品1.25mg/Kg实验组与阴性对照组存在显著差异，说明1.25mg/Kg实验组S和G_2/M期的细胞减少，细胞被阻止在G_0/G_1期，从而影响DNA的合成。1.25mg/Kg样品组与阴性对照组的p53蛋白表达量存在显著差异，说明凋亡通过p53正调控产生，阻止了DNA合成。Bcl-2蛋白表达量与阴性对照相比不存在显著差异，凋亡不是通过Bcl-2蛋白诱导产生。
综合利用质谱(GCT-MS、ESI)、氢谱(~1H-NMR)、碳谱(~(13)C-NMR)、二维谱图(DEPT)等现代波谱技术，对91018-1组分进行结构分析。经解析得知该化合物中文名称为粘噻唑。
In this paper, a total of100strains preserved by the Key Lab of Microbial Diversity Researchand Application of Hebei Province were used to screen anticancer drugs with tumor cell lines byMTT high throughout method.82%strains could produce high anticancer bioactive metaboliteagainst MCF-7tumor cell line. And six strains metabolite had good inhibition effect on MCF-7tumor cell and inhibited human embryo lung cells growth lightly at the same time.
For A549and HeLa cells, strain15had more inhibitive activity. The IC50of72h for A549and HeLa cells were6.66μg/mL and14.70μg/mL respectively. So it was selected for futherinvestigation.
The chromatography method had been studied and the suitable conditions had beendetermined and established. In the method, we used the Thermo ODS-2,5μm,250mm×4.6mmas HPLC column, acetonitrile: water=49:51as mobile phase.5components had been isolatedfrom the fermentation of strain15.
Compound15-1had significant inhibition on breast cancer MCF-7cells. The inhibition ratesof compound15-1on breast cancer MCF-7cells was71.73%, which the concentration was100μg/mL and there was good dose-effect relationship The inhibition rates of the other compoundswere not obvious.
91018-1had been isolated from the fermentation of strain91018which had anticanceractivity. The inhibition rates of compound91018-1on HeLa and MCF-7cells was69.19%and22.59%respectively when the concentration was100μg/mL. Compound91018-1had significantinhibition on breast cancer MCF-7cells.
The H_(22)tumor bearing mice was collected and the anticancer rate was calculated. Resultsshowed the inhibition rate of compound91018-1was48.7%when the dosage was1.25mg/kg.More or less was not better for the inhibition rate. The inhibition rate of positive control whichwas cyclophosphamide was81.3%. But characteristic features of mice were worse. One mousewas dead on the8th day. The1.25mg/kg group had better characteristic features such as behavioractivity, hair and mental status.
The expression of Bcl-2protein and p53protein in the tumor tissue were studied by theflow cytometry method. Results showed91018-1could induce tumor apoptosis by prevention ofcell cycle progression; Cells of S and G_2/M phase decreased. Cells were impeded at G_0/G_1phaseand thus affected DNA synthesis. The apoptosis was conducted by p53protein.
We had utilized modern spectroscopy testing technology such as ESI, GCT-MS,~1H-NMR,~(13)C-NMR and DEPT in order to confirm the structure of91018-1. After analyzing, we knew thatthe molecular weight of91018-1was487.68and the molecular formula was C_(25)H_(33)O_3N_3S_2. Thename of the compound is myxothiazol.
引文
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