【摘要】 通过细胞抑制试验研究桑黄子实体粗多糖与蛹虫草子实体粗多糖混合后的粗多糖对肺癌细胞A549的抑制作用,为利用药用真菌开发抗肿瘤药物提供试验依据。采用MTT法检测0.8 mg/m L的桑黄粗多糖和蛹虫草粗多糖对肺癌细胞A549增殖均有一定的抑制作用,作用48 h后的抑制率分别为32.95%、41.93%;将0.8 mg/m L桑黄粗多糖和0.8 mg/m L蛹虫草粗多糖按1∶1体积比混合的粗多糖则表现出协同增效抑制A549细胞增殖作用,抑制率可达54.71%。用流式细胞计数法测试分析的结果是:0.8 mg/m L蛹虫草粗多糖可影响A549细胞周期各时相的分布,而0.8 mg/m L桑黄粗多糖则对细胞周期无明显作用,但0.8 mg/m L混合粗多糖对A549细胞周期各时相分布的影响显著增强,G0/G1期细胞比率从75.39%±2.49%降低至64.43%±2.12%(P<0.01),S期细胞比率从16.61%±0.87%升高至27.97%±2.82%(P<0.01);混合粗多糖作用48 h后,A549细胞的晚期凋亡率从正常组的1.92%±0.55%提高至20.75%±0.24%,坏死细胞率由正常组的1.88%±0.43%提高至9.64%±0.42%,表现出明显的协同增效诱导A549细胞凋亡的作用。荧光定量PCR检测结果表明,A549细胞分别用0.8mg/m L的桑黄粗多糖和蛹虫草粗多糖及其混合粗多糖处理后,细胞内周期调控相关蛋白基因Cyclin A、CDK4的mRNA转录水平均显著下调,Cyclin D1、CDK2的mRNA转录水平显著上调,凋亡相关基因Bid、Bak、Cyt-C、Caspase3的mRNA转录水平也显著上调。研究结果提示桑黄粗多糖和蛹虫草粗多糖混合使用,具有协同增效抑制肺癌细胞A549增殖的作用,推测其通过调控细胞周期和细胞凋亡相关基因的表达,导致Cyt-C释放及启动caspase级联反应,从而诱导细胞凋亡。更多还原

【Abstract】 In this paper,inhibitive effect of the mixture of crude polysaccharides from fruit bodies of Phellinus spp. and Cordyceps sinensis on lung adenocarcinoma cell A549 were studied by cell inhibition test to evaluate the potential use of medical fungi as antitumor drugs. MTT assay showed that0. 8 mg/m L crude polysaccharides from Phellinus spp.and C. sinensis exhibited a certain inhibitive effect on A549 cell proliferation. The inhibitive rate was 32. 95%and 41. 93%,respectively at 48 h after treatment. Moreover,0. 8 mg/m L mixed crude polysaccharides from Phellinus spp. and C. sinensis at the volume ratio of 1 ∶ 1 could increase the inhibitive effect on A549 cells,and the inhibition rate was increased to 54. 71%. Flew cytometry exper-iment results showed that different cell cycle phase proportions of A549 cells were affected by 0. 8 mg/m L crude polysaccharides from C. sinensis,while 0. 8 mg/m L crude polysaccharides from Phellinus spp. had no obvious effect on the proportion of A549 cells in different phases. But,0. 8 mg/m L mixed crude polysaccharides had a significantly enhanced effect on different cell cycle phase proportions. The proportion of cells in G0/G1 phase was significantly decreased from75. 39%±2. 49% to 64. 43%±2. 12%,and that in S phase was increased from 16. 61%±0. 87% to 27. 97%±2. 82%( P<0. 01). After treated with mixed crude polysaccharides for 48 h,the rate of late apoptosis of A549 cells was increased from 1. 92%±0. 55% to 20. 75%±0. 24%( P<0. 01),and the rate of necrosis cells was increased from 1. 88%±0. 43% to9. 64%±0. 42%( P<0. 01). It was suggested that crude polysaccharides from Phellinus spp. and C. sinensis could synergistically induce A549 cell apoptosis. qRT-PCR results showed that mRNA transcriptional levels of cell cycle-related regulatory protein coding genes Cyclin A and CDK4 were significantly reduced,but those of Cyclin D1 and CDK2 were significantly increased in A549 cells treated with 0. 8 mg/m L crude polysaccharides from Phellinus spp. and C. sinensis and in A549 cells treated with mixed crude polysaccharides. Furthermore,mRNA transcriptional levels of apoptosis-related gene Bid,Bak,Cyt-C and Caspase3 were also significantly increased. These results indicated that the mixed crude polysaccharides from Phellinus spp. and C. sinensis could synergistically enhance the inhibition effect on A549 cell proliferation and induce cell apoptosis by regulating cell cycle-and apoptosis-related gene expression which resulted in the release of Cyt-C and initiation of caspase cascade reaction.更多还原