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目的:从土样中分离纯化粘细菌,对其进行鉴定与归类,以丰富粘细菌菌种资源,并对其进行抗肿瘤活性初步研究,为抗肿瘤药物开发奠定基础。方法:采用灭活大肠杆菌诱导法,从土样中分离纯化粘细菌,结合形态观察、生理生化特征及16S rRNA基因序列同源性分析进行菌株鉴定;向发酵液上清中加入XAD-16大孔吸附树脂提取发酵产物粗提物,CCK-8法进行体外抗肿瘤活性研究;RP-HPLC分离抗肿瘤活性组分,LC-MS/MS分析其分子质量。结果:分离并鉴定了STXZ77菌株,命名为Myxococcus stipitatus STXZ77。该菌株的发酵产物XAD-16树脂粗提物对小鼠黑色素瘤细胞B16、小鼠乳腺癌细胞4T1、人肝癌细胞SMMC-7721、人宫颈癌细胞He La、人结肠癌细胞SW480等多种肿瘤细胞具有较好的细胞毒性,作用24h的IC_(50)值分别为5.34μg/ml、13.50μg/ml、11.93μg/ml、28.70μg/ml、48.09μg/ml,而对正常细胞人脐静脉血管内皮细胞HUVEC的毒性较小,IC_(50)值为17.09μg/ml,小于B16、4T1及SMMC-7721的细胞毒性。RP-HPLC分离得到抗肿瘤活性组分AP-C,质谱分析其分子质量为422.99m/z。结论:从土样中分离得到粘细菌Myxococcus stipitatus STXZ77,从该菌中分离得到抗肿瘤活性组分AP-C,具有开发成抗肿瘤药物的潜在价值。
OBJECTIVE: To isolate and purify myxobacteria from soil samples and identify and classify them to enrich Mycobacterium species and to study their antitumor activity, which will lay the foundation for the development of anticancer drugs. Methods: The myxobacteria were isolated and purified from soil samples by inactivated E. coli induction. The morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis were used to identify the strains. XAD-16 was added to the supernatant of the fermentation broth The crude extract of the fermentation product was extracted by pore adsorption resin, and the anti-tumor activity was studied by CCK-8 method. The anti-tumor activity components were separated by RP-HPLC and analyzed by LC-MS / MS. Results: The STXZ77 strain was isolated and identified as Myxococcus stipitatus STXZ77. The crude extract of the XAD-16 resin of the strain was used for the treatment of mouse melanoma B16, mouse breast cancer 4T1, human hepatoma SMMC-7721, human cervical Hela, human colon cancer SW480 and other tumors The results showed that the IC50 values of cells for 24h were 5.34μg / ml, 13.50μg / ml, 11.93μg / ml, 28.70μg / ml and 48.09μg / ml respectively, Vascular endothelial cells HUVECs less toxic, IC 50 value was 17.09μg / ml, less than B16, 4T1 and SMMC-7721 cytotoxicity. The anti-tumor active component AP-C was isolated by RP-HPLC and its molecular mass was 422.99 m / z by mass spectrometry. Conclusion: Myxococcus stipitatus STXZ77 was isolated from soil samples. The anti-tumor active component AP-C was isolated from this strain and has the potential value of developing anti-tumor drugs.