摘要
癌症是一种发病率和死亡率均很高且呈上升趋势的疾病。由于其发病机制的复杂性,至今尚无完全治愈它的有效药物。微生物代谢产物是抗癌药物的丰富资源,目前全球使用的抗癌药物中,有50%是直接或间接来自微生物。土壤微生物种类齐全,数量多,从其代谢产物中发现各种活性物质的潜力巨大,是主要的微生物资源。
本实验采用MTT体外筛选法从350株土壤放线菌中初筛得到16株能明显抑制A549和HGC细胞生长的放线菌,又通过碘化丙啶(PI)荧光染色流式细胞仪技术复筛,结果显示:菌株1070抗肿瘤活性组分能致使32.07%的A549细胞出现亚二倍体峰。应用萃取、沉淀、大孔树脂初步分离活性产物,透析法测得该抗肿瘤活性成份的分子量大于10000道尔顿,全波长扫描、茚三酮显色和酸水解后纸层析结果表明:菌株1070抗肿瘤活性组分为多糖类物质,而非蛋白质类和核酸类物质。
MTT法检测菌株1070活性成分在各种浓度、不同时间对A549细胞的影响,测得其抗肿瘤活性物质对A549细胞的IC_(50)值为211μg/mL(72h);腹腔注射方式测定该活性物质的对小鼠的LD_(50)为5988mg/kg(2天)。扫描电镜观察到受药细胞的细胞膜连接复合物消失,细胞变圆;透射电镜观察到受药细胞胞浆内出现大量类似自噬体的小泡,且细胞核无明显改变,细胞膜完整,无内溶物外漏。罗丹明123荧光染色流式结果表明菌株1070活性物质能使A549细胞线粒体受到损伤,线粒体膜电位下降至43.72%,MDC(单丹磺酰尸胺)荧光染色流式仪检测出菌株1070活性物质能使A549细胞产生自噬体,比率为29.74%,荧光显微镜观察AO活细胞染色发现A549细胞胞浆内出现自噬体,Western-blot分别检测了LC3和Beclin-1蛋白表达情况,发现菌株1070活性物质不但可使LC3-Ⅰ型蛋白像LC3-Ⅱ型蛋白转化,而且使Beclin-1蛋白表达量显著增加。从上述实验结果初步判断,菌株1070活性物质是通过诱导A549细胞发生自噬来抑制肿瘤细胞增殖的。
通过对菌落、菌丝体形态的考察、生理生化特性的考察以及16S rDNA保守序列测定,初步判断菌株1070为Streptomyces capillispiralis。
Cancer is a disease with rising trend of high incidence and mortality rate. There are no specific medicines to cure cancer because of its complex pathogenesis. Microbial metabolites are the rich natural resource for development of anti-cancer drugs. Totally 50% anti-cancer drugs used through over the world are made from microorganism directly or indirectly. Soil microorganism is the most important microbe resource because of its complete type, large amount and large quantity all kinds of active materials potential metabolists.
In this research, 16 strains were firstly screened out from 350 soil actinomycetes which can inhibit the growth of A549 cell and HGC cell obviously by MTT assay. Then these strains were determined by PI immunofluorescence staining and flow cytometry. After the secondary screening, strain 1070 was obtained because its metabolites can inhibite 32.07% A549 cells growth. The active component of strain 1070 was isolated by extraction, precipitation, macroporous resin separation, and finally found its molecular weight over 10000 daltons. Some identification experiments, including full wavelength scanning, TLC stained with ninhydrin and acid hydrolysis, showed that the active component should be polysaeccharide, but not protein or nucleic acid.
The MTT assays showed that the IC50 value of anti-cancer component of strain 1070 was determined as 211μg/mL based on A549 cells growth inhibition(72h), and the intraperitoneal LD_(50) of anti-cancer component in mice was 5988mg/kg(2 days). The results of scanning electron microscopy indicated that the junctional complex between A549 cells disappeared and the cells grew rounded. Transmission electron microscopy results showed many autophagy vesicles observed in the cytoplasm, but no evident changes in cell nuclear and the cell membrane is intact with no cellular breakage. The results of Rho-damine123 staining followed by flow cytometry showed that A549 cell mitochondria was suffered damage and the mitochondrial membrane potential was reduced to 43.72%. The results of Monodansylcadaverine (MDC) staining followed by flow cytometry showed that the A549 cells were induced cell autophagy with the ratio of 29.74%. Acridine orange (AO) staining experiment showed that the autophagy bodies appeared in cytolymph under fluorescence microscope. Western-blot discoverd that levels of LC3-II and Beclin-1 significantly increased, while LC3- I reduced. All the results indicated that the mechanism of active component of strain 1070 was inducing A549 cells autophagy.
Based on colonial morphology, mycelial morphology, physiological and biochemical characteristics and 16S rDNA sequences, strain 1070 was preliminarily identified as Streptomyces capillispiralis.
引文
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