低水平活性氧对Ras癌基因诱导肝肿瘤发生的影响

Role of ROS in H-ras12V induced liver tumor

目的探讨活性氧(reactive oxygen species,ROS)在Ras癌基因诱导体内肝肿瘤发生中的作用。方法采用H-ras12V转基因肝癌小鼠模型,原位灌流法分离3个月龄雄鼠肝细胞和DCF-DA染色,流式细胞术检测肝细胞中的ROS水平;免疫荧光测定试剂盒检测7个月龄雄鼠肝组织ROS水平。对3和7个月龄雄鼠肝组织及肝肿瘤组织进行总蛋白提取,蛋白质印迹法检测ERK、p-ERK、p53、p21和Caspase-3的表达水平。结果流式细胞检测结果表明,3个月龄转基因雄鼠肝细胞中的ROS水平为7.81±0.53,比非转基因雄鼠肝细胞的1.00±0.16显著升高,t=21.31,P<0.001。免疫荧光检测表明,7个月龄转基因雄鼠肿瘤周围(P)组织中的ROS水平为18.25±4.41,比非转基因雄鼠肝(Wt)组织的2.42±0.61显著升高,t=6.16,P=0.004;比转基因雄鼠肿瘤(T)组织的3.18±1.75也显著升高,t=5.50,P=0.005。蛋白质印迹法检测表明,3个月龄转基因雄鼠肝组织中的p-ERK水平为5.36±0.32,比非转基因雄鼠肝组织的0.08±0.04显著升高,t=16.55,P=0.003。7个月龄转基因雄鼠肿瘤组织中p-ERK蛋白水平为22.09±6.57,比非转基因雄鼠肝组织的0.08±0.02显著升高,t=5.80,P=0.004;比转基因雄鼠肿瘤周围组织的8.60±3.23也显著升高,t=3.19,P=0.033;7个月龄转基因雄鼠肿瘤组织中p53蛋白水平为4.86±2.18,比非转基因雄鼠肝组织的39.23±13.43显著降低,t=-3.31,P=0.046;比转基因雄鼠肿瘤周围组织的31.09±7.60也显著降低,t=-3.32,P=0.029。7个月龄转基因雄鼠肿瘤组织中p21蛋白水平为4.38±3.28,比非转基因雄鼠肝组织的33.47±8.04显著降低,t=-5.95,P=0.010;比转基因雄鼠肿瘤周围组织的24.02±10.18也显著降低,t=-3.18,P=0.034。7个月龄转基因雄鼠肿瘤组织中的Caspase-3活化片段所占比例(17×103/35×103)为0.09±0.05,比非转基因雄鼠肝组织的0.22±0.04显著降低,t=-3.84,P=0.018;转基因雄鼠肿瘤周围组织的活化片段为0.10±0.05,比非转基因雄鼠肝组织的也显著降低,t=-3.68,P=0.021。结论在H-ras12V转基因小鼠肝肿瘤周围组织中,Erk信号通路的激活,诱导了ROS水平的升高,进而抑制了肝细胞的癌变。而在肝肿瘤中,Ras癌基因通过某些机制诱导了ROS水平的降低及p53/p21及Caspase-3凋亡通路的下调,进而促进了肝肿瘤的发生发展。

OBJECTIVE To investigate the role of reactive oxygen species（ROS）in Ras oncogene-induced liver tumor.METHORDS H-ras12 Vtransgenic hepatic tumor mice were explored.For 3-month-age male mice,the hepatocytes were isolated by in situ perfusion and stained by DCE-DA,and then the flow cytometry was performed to test the ROS levels in the hepatocytes.For 7-month-age male mice,the liver tissues were frozen sectioned and the ROS levels were detected by high quality oxidative stress ROS fluorescence assay kit.The total proteins were extracted from the liver tissues of 3-month-age and 7-month-age male mice.The expression levels of ERK,p-ERK,p53,p21 and Caspase-3in the liver tissue samples were detected by western blot.RESULTS Flow cytometry results showed that,the levels of ROS in hepatocytes of 3-month-age transgenic male mice were 7.81±0.53,significantly increased than that of non-transgenic male mice（1.00±0.16,t=21.31,P〈0.001）.Immunofluorescence showed that,the levels of ROS in 7-month-age transgenic male mice peri-tumor tissues were 18.25±4.41,significantly increased than that of non-transgenic male mice liver tissues（2.42±0.61,t=6.16,P=0.004）,and transgenic male mice tumor tissues（3.18±1.75,t=5.50,P=0.005）.Western blot showed that,the protein levels of p-ERK in 3-month-age transgenic male mice were 5.36±0.32,significantly increased than that of non-transgenic male mice liver（0.08±0.04,t=16.55,P=0.003）.The protein levels of p-ERK in 7-month-age male mice transgenic male mice tumor tissues were 22.09±6.57,significantly increased than that of non-transgenic male mice liver（0.08±0.02,t=5.80,P=0.004）,and P（8.60±3.23,t=3.19,P=0.033）.The protein levels of p53 in 7-month-age male mice transgenic male mice tumor tissues were 4.86±2.18,significantly decreased than that of non-transgenic male mice liver（39.23±13.43,t=-3.31,P=0.046）,and transgenic male mice peri-tumor（31.09±7.60,t=-3.32,P=0.029）.The protein levels of p21 in 7-month-age male mice transgenic male mice tumor tissues were 4.38±3.28,significantly decreased than that of non-transgenic male mice liver（33.47±8.04,t=-5.95,P=0.010）,and transgenic male mice peri-tumor（24.02±10.18,t=-3.18,P=0.034）.The proportion of cleavage fragment of Caspase-3（17×103/35×103）in transgenic male mice tumor tissues were 0.09±0.05,significantly decreased than that of non-transgenic male mice liver（0.22±0.04,t=-3.84,P=0.018）;the proportion of cleavage fragment of Caspase-3（17×10^3/35×10^3）in transgenic male mice peri-tumor tissues（0.10±0.05）were significantly decreased than that of non-transgenic male mice liver（t=-3.68,P=0.021）.CONCLUSIONS In the hepatic peri-tumor tissues of H-ras12 Vtransgenic male mice,activation of Erk pathway elevates the levels of ROS which blocks the tumorigenesis.However,in hepatic tumors,Ras oncogene down-regulates the levels of ROS and the apoptosis pathway of p53/p21 and Caspase-3by unkown mechanisms to promote the tumorigenesis and development.