CAJ | 학술논문

目的通过过氧化氢诱导肝细胞及肝癌细胞产生氧化应激,探讨氧化应激与Hippo信号通路中核心蛋白Yes相关蛋白(YAP)的相关性.方法利用过氧化氢诱导构建氧化应激模型,用200 μmol/L过氧化氢作用于正常人肝细胞L02与肝癌细胞MHCC97L 6 h,Western blot检测YAP蛋白表达的差异,反转录-聚合酶链反应(RT-PCR)检测YAP基因mRNA表达差异,流式细胞术检测L02细胞周期的变化,比较实验组和对照组差异.结果 200 μmol/L的过氧化氢作用于L02细胞及MHCC97L细胞6h后,与对照组比较,实验组两种细胞YAP蛋白表达明显升高.L02细胞中YAP基因mRNA表达水平上调479.5％,差异有统计学意义(P＜0.01),MHCC97L细胞上调4.7％,差异无统计学意义(P＞0.05).流式细胞术检测细胞周期,L02细胞实验组与对照组比较S期细胞比例显著升高[(46.40±4.88)％比(28.61±1.01)％],差异有统计学意义(P＜0.01),G1期[(45.21±9.45)％比(59.59±2.44)％和G2期(8.42±5.14)％比(11.70±3.35)％]细胞分布改变不明显,差异无统计学意义(P＞0.05).结论在氧化应激状态的L02细胞及MHCC97L细胞中,Hippo信号通路存在调控紊乱,核心蛋白YAP表达上调.其中L02细胞存在转录水平的调控机制,MHCC97L细胞存在转录后水平的调控机制.氧化应激可以促进L02细胞增殖,YAP蛋白的表达升高可能是其中的原因之一.
목적 통과과양화경유도간세포급간암세포산생양화응격,탐토양화응격여Hippo신호통로중핵심단백Yes상관단백(YAP)적상관성.방법 이용과양화경유도구건양화응격모형,용200 μmol/L과양화경작용우정상인간세포L02여간암세포MHCC97L 6 h,Western blot검측YAP단백표체적차이,반전록-취합매련반응(RT-PCR)검측YAP기인mRNA표체차이,류식세포술검측L02세포주기적변화,비교실험조화대조조차이.결과 200 μmol/L적과양화경작용우L02세포급MHCC97L세포6h후,여대조조비교,실험조량충세포YAP단백표체명현승고.L02세포중YAP기인mRNA표체수평상조479.5％,차이유통계학의의(P＜0.01),MHCC97L세포상조4.7％,차이무통계학의의(P＞0.05).류식세포술검측세포주기,L02세포실험조여대조조비교S기세포비례현저승고[(46.40±4.88)％비(28.61±1.01)％],차이유통계학의의(P＜0.01),G1기[(45.21±9.45)％비(59.59±2.44)％화G2기(8.42±5.14)％비(11.70±3.35)％]세포분포개변불명현,차이무통계학의의(P＞0.05).결론 재양화응격상태적L02세포급MHCC97L세포중,Hippo신호통로존재조공문란,핵심단백YAP표체상조.기중L02세포존재전록수평적조공궤제,MHCC97L세포존재전록후수평적조공궤제.양화응격가이촉진L02세포증식,YAP단백적표체승고가능시기중적원인지일.
Objective To investigate the correlation between H2O2-induced oxidative stress of L02 cells and MHCC97L cells with Yes-associated protein (YAP) expression.Methods L02 cells and MHCC97L cells were exposed to 200 μmol/L H2O2 to establish the oxidative stress model.The expression of YAP protein and mRNA was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR),respectively.The cell cycles were examined by flow cytometry.Results After treated with 200 μ mol/L H2O2 for 6 h,the expression of YAP protein was up-regulated in both L02 cells and MHCC97L cells as compared with the control group.The reverse transcriptase-PCR results showed that the YAP mRNA levels were significant increased by 479.5％ (P ＜0.01) in L02 cells but had no significant change (increased by 4.7％,P ＞0.05) in MHCC97L cells.The flow cytometry results indicated more L02 cells went into S phase [(46.40 ± 4.88) ％ vs.(28.61 ± 1.01) ％,P ＜ 0.01] and ther were no significant changes in G1,and G2 phases [(45.21 ± 9.45) ％ vs.(59.59 ± 2.44) ％,and (8.42 ± 5.14) ％ vs.(11.70 ± 3.35) ％,P ＞ 0.05].Conclusion Oxidative stress can promote YAP protein expression in L02 cells and MHCC97L cells,and promote proliferation of L02 cells.