樟芝皿培菌丝提取物体外对SPCA-1肺癌细胞增殖的抑制作用  

Anti-proliferation Effect of Extracts from Dish-Cultivated T.Camphoratus Mycelia on SPCA-1 Lung Cancer Cells

作　　者：龙云凤[1] 吴莹莹[2] 贾薇[2] 杨海芮 白旭 张劲松[2] 杨焱[2] 张赫男[2] 栾军[1] 高玲[1] 王毅谦[1] 汪雯翰[2] LONG Yunfeng;WU Yingying;JIA Wei;YANG Hairui;BAI Xu;ZHANG Jingsong;YANG Yan;ZHANG Henan;LUAN Jun;GAO Ling;WANG Yiqian;WANG Wenhan(Animal, Plant and Food Inspection Center, Jiangsu Entry Exit Inspection and Quarantine Bureau, Nanjing 210019 ,China;National Engineering Research Center of Edible Fungi, Key Laboratory of Applied Mycological Resources and Utilization of Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding,Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences,Shanghai 201403 , China;WuXi AppTec, Shanghai,200131;College of Life Sciences,Shihezi University,Shihezi 832003 China)

机构地区：[1]江苏出入境检验检疫局动植物与食品检测中心,江苏南京210019 [2]上海市农业科学院食用菌研究所,农业部南方食用菌资源利用重点实验室,国家食用菌工程技术研究中心,国家食用菌加工技术研发分中心,上海市农业遗传育种重点开放实验室,上海201403 [3]药明康德新药开发有限公司,上海200131 [4]石河子大学生命科学学院,新疆石河子832000

出　　处：《食用菌学报》2019年第2期88-96,共9页Acta Edulis Fungi

基　　金：江苏出入境检验检疫局科技计划项目(2018KJ33)

摘　　要：利用细胞克隆实验、划痕实验和alamarBlue^TM细胞活力测定实验评价樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相对SPCA-1非小细胞肺癌的体外抑制作用,并进一步利用流式细胞术Annexin V-FITC(fluorescein isothiocyanate)/PI(propidium iodide)双染测定细胞早期凋亡率、流式细胞术2',7'-二氯荧光素二乙酸酯(2',7'-dichlorofluorescindiacetate,H2DCFDA)单染测定ROS(reactive oxygen species)释放量、流式细胞术PI单染测定细胞周期变化和ELISA(enzyme-linked immunosorbent assay)检测细胞凋亡相关蛋白Caspase-3、p53和PARP的表达,探讨樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相抑制SPCA-1增殖的作用机理。研究结果表明:樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相均能抑制SPCA-1细胞克隆的形成、迁移和增殖,石油醚萃取相的活性明显好于氯仿萃取相;石油醚萃取相通过促进SPCA-1释放ROS来诱发线粒体依赖性早期凋亡的发生,并使促凋亡蛋白Caspase-3和p53表达上升,同时抑制蛋白PARP表达,从而发挥其抑制SPCA-1肺癌细胞增殖的作用;而氯仿萃取相主要通过将SPCA-1细胞阻滞在S期抑制其增殖。Colony formation method, wound-healing method and alamarBlue^TM cell viability assay were used to evaluate in vitro anti tumor effects of petroleum ether extract and chloroform extract from dish-cultivated T. camphoratus mycelia on SPCA-1 non-small cell lung cancer cells. Flow cytometry Annexin V-FITC/PI double staining method was used to determine early apoptotic rate;flow cytometry 2',7'dichlorofluorescindiacetate(H2DCFDA) single staining method was used to determine ROS (reactive oxygen species) release;flow cytometry PI single staining method was applied to determine cell cycle changes;and ELISA method was used to determine expression of apoptosis-related proteins Caspase-3, p53 and PARP, hence to explore the anti-proliferation mechanism of extracts from dish-cultivated T.c amphoratus mycelia on SPCA-1 lung cancer cells. The results showed that both petroleum ether extract and chloroform extract inhibited the colony formation, migration and proliferation of SPCA-1 cells. The activity of petroleum ether extract was significantly better than that of chloroform extract. The petroleum ether extract induced mitochondria-dependent early apoptosis by promoting the release of ROS from SPCA-1, which led to an increase in the activity of the pro-apoptotic proteins Caspase-3 and p53 and inhibition of PARP protein, thereby exerting its anti-tumor activity. The chloroform extract might exert its anti-tumor activity mainly by arresting SPCA-1 cells in S phase.

关 键 词：克隆形成 迁移 活性氧 凋亡 细胞周期 CASPASE-3 p53 PARP 

分 类 号：R285[医药卫生—中药学]