CAJ | 학술논문

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.
목적 탐토감성성섬유세포생장인자(bFGF)여신호전도화전록활화인자3(STAT3)재인효질류세포조망중적관계급가능적작용궤제.방법 이인효질모세포류U87세포주화U251세포주위연구대상,분위정상대조조、공재체조、실험조,분별구건휴대파향bFGF화STAT3소분자간우RNA적중조만병독LV-bFGF-siRNA화LV-STAT3-siRNA,진행만병독전염.작용48 h후가입소분자억제제AG490 50 μmol/L、LY294002 20 μmol/L,선택성조단JAK、PI3 K/Akt통로,작용24、48、72 h후,응용Western blot법검측bFGF、STAT3、pSTAT3(Tyr705)화pSTAT3(Ser727)적표체정황,용류식세포의、단백심편、격광공초현미경분별검측세포조망정황、조망상관단백표체급선립체막전위적변화.다조간균수비교채용단인소방차분석.결과 Western blot검측결과현시,JAK통로조단후p-STAT3(Tyr705)적표체량수시간증가이강저,PI3 K/Akt통로조단후p-STAT3(Ser727)적표체량수시간증가이강저;류식세포의검측결과현시,U87세포주화U251세포주중정상대조조、공재체조、실험조세포적조망비례분별위17.97％±0.24％、18.26％±0.88％、46.57％±1.63％화15.94％±1.18％、16.88％±0.17％、39.34％±0.87％.정상대조조여공재체조상비차이무통계학의의(P치균＞0.05),여실험조상비,차이균유통계학의의(F=697.41、729.58,P치균＜0.05).단백심편결과현시,실험조화공재체조상비,Bad、Caspase3、Cytochrome C、p27적표체량증가,XIAP적표체량하강.격광공초현미경하가견여정상조화공재체조상비,실험조세포선립체막전위강저.결론 인효질모세포류세포중bFGF가통과작용우pSTAT3(Tyr705)래영향STAT3적린산화수평;조단bFGF/STAT3신호전도통로가유도효질류세포선립체도경적세포조망.
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 membrane potential reduced significantly in the experimental group compared with the normal control group and the mock group.Conclusions bFGF mainly interacts with STAT3 tyrosine site-pSTAT3 (Tyr705) to influence the level of STAT3 phosphorylation; blocking bFGF/STAT3 signaling pathway can induce glioma cell apoptosis through mitochondrial pathway.