腺病毒介导的东亚钳蝎氯毒素对人胶质瘤U251细胞的作用  

Effect of adenovirus-mediated recombinant Buthus martensii Karsch chloride toxin artifact on human glioma U251 cells

作　　者：陈良崇 胡涛 周浪浪 黄瑞[2] 刘宝来 郭慧敏[2] 陈胜利 Chen Liangchong;Hu Tao;Zhou Langlang;Huang Rui;Liu Baolai;Guo Huimin;Chen Shengli(Department of Neurosurgery,Shanxi Provincial People's Hospital,the Affiliated People's Hospital of Shanxi Medical University,Taiyuan 030012,China;Department of Clinical Laboratory,Children's Hospital of Shanxi Province,Taiyuan 030002,China)

机构地区：[1]山西省人民医院山西医科大学附属人民医院神经外科,大原030012 [2]山西省儿童医院检验科,大原030002

出　　处：《肿瘤研究与临床》2021年第4期264-269,共6页Cancer Research and Clinic

基　　金：山西省自然科学基金(2015011120)。

摘　　要：目的探讨腺病毒介导的人工合成东亚钳蝎氯毒素(Ad-rBmK CTa)对人胶质瘤U251细胞的抑制作用及其相关机制。方法设置3.5×109、7.0×109、3.5×1010pfu/ml三个效价梯度Ad-rBmK CTa组,分别作用于U251细胞24、48、72 h,同时设置空白对照组(不加入细胞和Ad-rBmK CTa)、阴性对照组(只加入U251细胞)。通过激光共聚焦荧光显微镜观察病毒感染情况;采用四甲基偶氮唑盐(MTT)法检测各组细胞增殖情况;采用流式细胞术检测各组细胞周期及细胞凋亡;采用蛋白质印迹法检测凋亡相关蛋白bax、bcl-2和caspase-3表达情况。结果 Ad-rBmK CTa作用于U251细胞24 h后感染效率达90%以上。随着Ad-rBmK CTa效价升高,作用相同时间的U251细胞增殖抑制率逐渐升高(均P<0.01);随着时间延长,同一效价Ad-rBmK CTa作用的U251细胞增殖抑制率亦逐渐升高(均P<0.01)。7.0×10^(9) pfu/ml Ad-rBmK CTa作用于U251细胞48 h后,G0/G1期细胞比例为(40.7±0.8)%,S期和G2期细胞比例分别为(35.7±0.6)%、(23.6±1.4)%,差异具有统计学意义(F=225.119,P<0.01)。7.0×10^(9) pfu/ml Ad-rBmK CTa作用于U251细胞24、48、72 h时,细胞凋亡率分别为(7.4±1.4)%、(19.2±1.7)%和(22.3±1.7)%,差异有统计学意义(F=49.470,P<0.01)。7.0×10^(9) pfu/ml Ad-rBmK CTa作用于U251细胞48 h后,与阴性对照组相比,bax、caspase-3蛋白的表达水平升高,bcl-2蛋白表达水平下降。结论 Ad-rBmK CTa可能作用于DNA损伤诱导的G1/S检测点,使细胞周期停滞于G0/G1期,从而抑制U251细胞的体外增殖,但其诱导U251细胞凋亡的作用并不明显。其机制可能与氯离子-通道直接或间接受到抑制相关。Objective To investigate the inhibitory effect of adenovirus-mediated recombinant Buthus martensii Karsch chloride toxin artifact(Ad-rBmK CTa)on human glioma U251 cells and its related mechanisms.Methods Groups of 3 titer gradients of 3.5×10^(9),7.0×10^(9) and 3.5×10^(10) pfu/ml Ad-rBmK CTa were set up and applied to U251 cells for 24,48 and 72 h,and a blank control group(no cells and Ad-rBmK CTa were added)and a negative control group(only U251 cells were added)were set up at the same time.The virus infection status was observed by laser confocal fluorescence microscopy.The cell proliferation in each group was detected by methyl thiazolyl tetrazolium(MTT)assay.The cell cycle and apoptosis in each group were detected by flow cytometry.The expressions of apoptosis-related proteins bax,bcl-2 and caspase-3 were detected by Western blot.Results The infection rate of Ad-rBmK CTa was over 90%after acting on U251 cells for 24 h.As the titer of Ad-rBmK CTa increased,the proliferation inhibition rate of U251 cells treated for the same hours gradually increased(all P<0.01);with the extension of time,the proliferation inhibition rate of U251 cells treated with the same titer of Ad-rBmK CTa also gradually increased(all P<0.01).After 7.0×10^(9) pfu/ml Ad-rBmK CTa acted on U251 cells for 48 h,the proportion of cells in G0/G1 phase was(40.7±0.8)%,and cells in S phase and G2 phase accounted for(35.7±0.6)%and(23.6±1.4)%,and the difference was statistically significant(F=225.119,P<0.01).When 7.0×10^(9) pfu/ml Ad-rBmK CTa acted on U251 cells for 24,48 and 72 h,the apoptosis rates were(7.4±1.4)%,(19.2±1.7)%and(22.3±1.7)%(F=49.470,P<0.01).After 7.0×10^(9) pfu/ml Ad-rBmK CTa acted on U251 cells for 48 h,compared with the negative control group,the expressions of bax and caspase-3 proteins increased,and the expressions of bcl-2 decreased.Conclusions Ad-rBmK CTa may act on the DNA damage-induced G1/S detection site to arrest the cell cycle in G0/G1 phase,thus inhibiting the proliferation of U251 cells in vitro.However,its in

关 键 词：神经胶质瘤 腺病毒科 蝎毒素 细胞周期 细胞凋亡 氯化物通道 

分 类 号：R739.4[医药卫生—肿瘤]