PMA诱导Hela细胞ROS升高机制研究  被引量:2

Mechanism of ROS increasing induced by PMA in Hela cells

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作  者:李树林 王超[1] 安雅男 李燕 王雪艳[2] 唐旭东[2] 于录[1] LI Shu-lin;WANG Chao;AN Ya-nan;LI Yan;WANG Xue-yan;TANG Xu-dong;YU Lu(Key Laboratory for Zoonosis Research (Ministry of Education ), Institute of Zoonosis , Jilin University, Changchun 130062, P. R. China;Key Laboratory for New Drug Research of TCM,Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, P. R. China)

机构地区:[1]吉林大学人兽共患病研究所人兽共患病研究教育部重点实验室,吉林长春130062 [2]深圳清华大学研究院新药研发重点实验室,广东深圳518057

出  处:《中华肿瘤防治杂志》2018年第9期622-627,共6页Chinese Journal of Cancer Prevention and Treatment

基  金:吉林省科技发展计划(20150101108JC);吉林省教育厅项目(2016444);深圳市科学技术创新委员会项(JSGG2016030-1100442775)

摘  要:目的活性氧簇(reactive oxygen species,ROS)升高是癌症转移的主要原因之一,ROS已成为一种新的抗癌靶点,但其形成机制尚未完全阐明。本研究以佛波酯(phorbol-12-myristate-13-acetate,PMA)诱导Hela细胞ROS升高为模型,探讨Hela细胞ROS升高的相关机制。方法 500ng/mL的PMA诱导Hela细胞0.5h,荧光酶标仪检测Hela细胞经过PMA诱导后的ROS产量,蛋白质印迹法检测Hela细胞经过PMA诱导后的HIF-1α、Rac2和NOX2蛋白的表达量。结果 500ng/mL PMA诱导Hela细胞0.5、1、2和4h,与空白对照组比较,P值分别为<0.001、0.055、0.125和0.135,表明0.5h为PMA诱导Hela细胞ROS升高的最佳诱导条件。500ng/mL PMA诱导Hela细胞0.5h后,诱导组中HIF-1α、Rac2和NOX2蛋白条带灰度平均值分别为15 833.33±63.13、20 448.33±78.65和21 840.33±123.91,均高于空白组的9 853.33±124.13、9 782.00±93.62和10 638.33±106.57,均P<0.001,表明PMA诱导了HIF-1α、Rac2和NOX2蛋白的高表达。荧光酶标仪检测Hela细胞ROS结果显示,echinomycin组、NSC23766组和DPI组与PMA组比较,P值分别为<0.001、0.003和<0.001,表明HIF-1α、Rac2和NOX2抑制剂均抑制了Hela细胞中PMA诱导的ROS升高。蛋白质印迹法检测结果显示,空白组HIF-1α、Rac2和NOX2灰度平均值分别为13 571.00±287.68、10 495.67±253.72和17 403.33±162.87,PMA组分别为22 946.33±67.10、30 578.67±251.11和30 582.00±88.39,NSC23766组分别为22 629.67±445.26、3 919.00±41.68和22 618.67±161.51,echinomycin组分别为16 691.33±9.504、15 424.00±315.05和14 899.00±71.55,DPI组分别为22 970.00±714.88、30 120.33±555.05和5 787.00±12.29。echinomycin组与PMA组比较,均P<0.001,表明echinomycin抑制了PMA诱导的HIF-1α、Rac2和NOX2蛋白的高表达;NSC23766组与PMA组比较,P值分别为0.354、<0.001和<0.001,表明NSC23766抑制了PMA诱导的Rac2和NOX2蛋白的高表达,但没有抑制HIF-1α蛋白的高表达;DPI组与PMA组比较,P值分别为0.944、0.117和<0.001,表明DPI抑制了PMA诱导的NOX2OBJECTIVE Increased ROS is one of the major cause of cancer metastasis.ROS has become a new anticancer target,yet its mechanism has not been fully elucidated.This study used PMA(Phorbol-12-myristate-13-acetate)to induce the increase of ROS(reactive oxygen species)in Hela cells,and investigate the mechanism of the increase of ROS in Hela cells.METHODS The Hela cells were treated with 500 ng/mL PMA for 0.5 h.The production of ROS induced by PMA was detected by fluorescence microtiter plate reader.The expression of HIF-1α,Rac2 and NOX2 protein in Hela cells induced by PMA was detected by Western blotting.Image J software was used to calculate the results.RESULTS After Hela cells were induced by 500 ng/mL PMA for 0.5 h,1 h,2 hand 4 h,the ROS level in Hela cells was detected by fluorescence microplate reader.Compared with the blank control group,the Pvalues were〈0.001,0.055,0.125 and 0.135.The results showed that the optimal induction time of ROS increase induced by PMA in Hela cells was 0.5 h.Hela cells were induced by 500 ng/mL PMA for 0.5 h.Western blot results showed that the mean gray scales of the HIF-1α,Rac2 and NOX2 bands in the induced group were 15 833.33±63.13,20 448.33±78.65 and 21 840.33±123.91,respectively.The blank groups were 9 853.33±124.13,9 782.00±93.62 and 10 638.33±106.57,respectively.The average gray value of HIF-1α,Rac2 and NOX2 protein in the induction group was higher than that of the blank group.The average gray levels of HIF-1α,Rac2 and NOX2 in the induced group were compared with the blank group(P〈0.001),indicating that PMA induced high expression of HIF-1α,Rac2 and NOX2 proteins in Hela cells.ROS levels in Hela cells were detected by fluorescence microplate reader.The Pvalues in the echinomycin group,NSC23766 group and DPI group were〈0.001,0.003 and〈 0.001 respectively.The results showed that HIF-1α,Rac2 and NOX2 inhibitors significantly inhibited ROS increase from PMA-induced in Hela cells.Western blot results showed that the average gray levels of HIF-1α,

关 键 词:肿瘤 佛波酯 HELA细胞 活性氧簇 HIF-1Α Rac2 NOX2 印迹法 蛋白质 

分 类 号:R73-3[医药卫生—肿瘤]

 

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