眼镜蛇毒L-氨基酸氧化酶对人膀胱癌ECV304细胞增殖和细胞周期的影响及机制  

Effects of Naja atra L-amino acid oxidase on the proliferation and cell cycle of human bladder carcinoma ECV304 cells

作　　者：齐元麟[1] 张明芳[1,2] 赵蓉[1] 张旭[1] 吴玲珊[1] 林建银[1] 

机构地区：[1]福建医科大学基础医学院分子医学研究中心消化道恶性肿瘤教育部重点实验室,福建福州350004 [2]福建医科大学生理学与病理生理学系,福建福州350004

出　　处：《中国药理学通报》2011年第12期1660-1665,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 30371747);福建省教育厅科技重点资助项目(No JA10130);福建医科大学青年科学基金资助项目(No FJGXQ04011);福建医科大学大学生创新性实验计划资助项目(No C10050)

摘　　要：目的研究眼镜蛇毒L-氨基酸氧化酶(Naja atra L-a-mino acid oxidase,NA-LAAO)对人膀胱癌细胞株ECV304的生长抑制作用,探讨其作用机制。方法 CCK-8法测定NA-LAAO的细胞毒性及对细胞增殖抑制能力;流式细胞术分析NA-LAAO对ECV304细胞周期的影响;实时定量PCR和Western blot分析细胞周期相关基因mRNA和蛋白质表达。结果 NA-LAAO对ECV304细胞有强烈抑制作用,且呈剂量-效应和时间-效应关系。NA-LAAO处理6、12、24 h对ECV304的IC50分别为(1.54±0.23)、(1.09±0.15)和(0.48±0.14)mg.L-1;细胞生长曲线显示,0.156 mg.L-1的NA-LAAO作用4 d可明显抑制ECV304细胞增殖;0.313 mg.L-1从d 2起就有明显效果;0.625 mg.L-1处理后细胞多数死亡;流式细胞术分析提示NA-LAAO可使ECV304细胞阻滞在G1期,并呈量效关系;实时定量PCR结果显示,NA-LAAO可下调Cyclin A2、B1、D1、E1、CDK2、CDK6、Survivin、Skp2,上调p21、p16、p27的表达,Western blot结果显示NA-LAAO可下调Cyclin A、CDK2、CDK6,上调p21,与实时PCR结果一致。结论 NA-LAAO可通过调节细胞周期相关分子,使ECV304细胞阻滞在G1期,抑制细胞增殖。Aim To investigate the effects and the molecular mechanisms of Naja atra L-amino acid oxidase（NA-LAAO） on the proliferation and cell cycle of human bladder carcinoma ECV304 cells.Methods ECV304 cells were exposed to different concentrations of NA-LAAO and the cytotoxicity was measured by CCK-8 method.The effects of NA-LAAO on cell cycle were examined by a propidium iodide staining in flow cytometry.The mRNA or protein expressions of cell cycle regulators were determined by real-time PCR or Western blot,respectively.Results NA-LAAO exhibited cytotoxicity in ECV304 in a dose-and time-dependent manner.The half maximal inhibitory concentration（IC50） values of NA-LAAO were（1.54±0.23）,（1.09±0.15） and（0.48±0.14） mg·L-1 in ECV304 cells at 6 h,12 h and 24 h,respectively.At concentrations lower than 0.625 mg·L-1,NA-LAAO did not kill the ECV304 cells but inhibited cell proliferation.Four days＇exposure to NA-LAAO of 0.156 mg·L-1 or two days＇exposure to NA-LAAO of 0.313 mg·L-1 caused a significant decrease of cell growth.Analysis of ECV304 cell cycle by flow cytometry method indicated that NA-LAAO could increase the G1-phase cells and block the cell cycle in G1-phase.Real-time PCR analysis indicated NA-LAAO up-regulated the mRNA expression of p21,p16,p27 whereas down-regulated the expression of cyclin A2,cyclin B1,cyclin D1,cyclin E1,CDK2,CDK6,Survivin and Skp2.Western blot analysis indicated NA-LAAO up-regulated p21 and down-regulated cyclin A,CDK2 and CDK6.These effects might contribute to the blockade of cell cycle.Conclusion NA-LAAO inhibits cell proliferation by blocking the cell cycle in G1-phase.

关 键 词：蛇毒 中华眼镜蛇 L-氨基酸氧化酶 细胞增殖 细胞周期 膀胱癌 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]