碱性成纤维细胞生长因子与信号转导和转录活化因子3在人胶质瘤细胞凋亡中的关系  

A preliminary study about the interaction between basic fibroblast growth factor and signal transducer and activator of transcription 3 in glioma apoptosis

作　　者：冯学泉[1] 吴静超[2] 徐新女[3] 刘宏胜[3] 刘俊[2] 李家林[2] 张飚[2] 王金环[2] 

机构地区：[1]天津市第一中心医院神经外科,300192 [2]天津市环湖医院 [3]卫生部危重病急救医学重点实验室

出　　处：《中华外科杂志》2014年第12期939-944,共6页Chinese Journal of Surgery

基　　金：国家自然科学基金青年科学基金资助项目（81101911）;天津市科技计划项目（12ZCDZSY11700）;天津市应用基础与前沿技术研究计划（14JCZDJC35600）

摘　　要：目的 探讨碱性成纤维细胞生长因子（bFGF）与信号转导和转录活化因子3（STAT3）在人胶质瘤细胞凋亡中的关系及可能的作用机制.方法 以人胶质母细胞瘤U87细胞株和U251细胞株为研究对象,分为正常对照组、空载体组、实验组,分别构建携带靶向bFGF和STAT3小分子干扰RNA的重组慢病毒LV-bFGF-siRNA和LV-STAT3-siRNA,进行慢病毒转染.作用48 h后加入小分子抑制剂AG490 50 μmol/L、LY294002 20 μmol/L,选择性阻断JAK、PI3 K/Akt通路,作用24、48、72 h后,应用Western blot法检测bFGF、STAT3、pSTAT3（Tyr705）和pSTAT3（Ser727）的表达情况,用流式细胞仪、蛋白芯片、激光共焦显微镜分别检测细胞凋亡情况、凋亡相关蛋白表达及线粒体膜电位的变化.多组间均数比较采用单因素方差分析.结果 Western blot检测结果显示,JAK通路阻断后p-STAT3（Tyr705）的表达量随时间增加而降低,PI3 K/Akt通路阻断后p-STAT3（Ser727）的表达量随时间增加而降低;流式细胞仪检测结果显示,U87细胞株和U251细胞株中正常对照组、空载体组、实验组细胞的凋亡比例分别为17.97％±0.24％、18.26％±0.88％、46.57％±1.63％和15.94％±1.18％、16.88％±0.17％、39.34％±0.87％.正常对照组与空载体组相比差异无统计学意义（P值均＞0.05）,与实验组相比,差异均有统计学意义（F=697.41、729.58,P值均＜0.05）.蛋白芯片结果显示,实验组和空载体组相比,Bad、Caspase3、Cytochrome C、p27的表达量增加,XIAP的表达量下降.激光共焦显微镜下可见与正常组和空载体组相比,实验组细胞线粒体膜电位降低.结论 人胶质母细胞瘤细胞中bFGF可通过作用于pSTAT3（Tyr705）来影响STAT3的磷酸化水平;阻断bFGF/STAT3信号转导通路可诱导胶质瘤细胞线粒体途径的细胞凋亡.Objective To study the relationship of basic fibroblast growth factor （bFGF） and signal transducer and activator of transcription 3 （STAT3） in glioma apoptosis and possible mechanisms of its interaction.Methods Two glioblastomamultiforme （GBM） cell lines：U87 （wild-type p53） and U251 （mutant p53） were used in this study and divided into normal control group,mock group and experiment group.Small interfering RNA-carried recombinant lentivirus,LV-bFGFsiRNA and LV-STAT3siRNA,targeting bFGF and STAT3 were constructed respectively.After 48 hours of lentivirus transfection,small molecular inhibitors were used to block specific signaling pathways,AG490 20 μmol/L blocking JAK,LY294002 20 μmol/L blocking PI3K/Akt pathways for 24 hours,48 hours and 72 hours,respectively.Then,apoptosis,changes in apoptosis-related proteins and mitochondrial membrane potential were detected through the methods of flow cytometry,protein chip and confocal microscopy,respectively.Groups were compared using single factor analysis of variance （One-way ANOVA）.Results Western blot results revealed the levels of Tyr705 and Ser727 phosphorylationin reduced in a time dependent manner by blocking JAK and PI3K/Akt pathway respectively.The results of flow cytometry showed that the apoptosis rate in normal control group,mock group,experiment group were 17.97％ ±0.24％,18.26％ ±0.88％,46.57％ ± 1.63％ in U87 cells and 15.94％ ± 1.18％,16.88％ ± 0.17％,39.34％ ± 0.87％ in U251 cells,respectively.There was no statistically significant change between the normal control group and the mock group（P ＞ 0.05）,while when compared with the experiment group,both group showed statistically significant difference （F =697.41,729.58,both P ＜ O.05）.The results of protein chip demonstrated that protein expression of Bad,Caspase3,Cytochrome C,p27 were higher and XIAP was lower in the experiment group compared with the normal control group and mock group.Also,confocal microscopy could detect apoptosis and mitochondrial m

关 键 词：成纤维细胞生长因子 STAT转录因子类 神经胶质瘤 细胞凋亡 

分 类 号：R739.4[医药卫生—肿瘤]