p63不同异构体表达模式对细胞周期特异性毒物诱导的肝癌细胞生长的影响

目的研究肝癌细胞中p63不同异构体表达模式对该细胞阿霉素敏感性的影响。方法用0（溶剂对照）、0.08、0.16、0.32、0.64和1.28μmol/L阿霉素处理对数生长期的p53基因缺失Hep3B细胞,采用MTT比色法检测Hep3B细胞的存活率变化,采用流式细胞术分析Hep3B细胞凋亡率变化,通过彗星实验评价Hep3B细胞DNA损伤水平,采用Western blott方法检测Hep3B细胞中TAp63、DNp63和Cytochrome c的蛋白水平。结果阿霉素处理后的Hep3B细胞存活率依次为89%、71%、54%、38%和22%;凋亡率依次为16%、28%、42%、56%和78%;Hep3B细胞DNA损伤程度依次为2.9、5.1、7.2、9.4和12.1;上调了TAp63和Cytochrome c蛋白表达水平,而DNp63蛋白表达水平则无显著变化,使得TAp63/DNp63比值随之显著升高。结论 TAp63/DNp63比值的增加可显著提高Hep3B细胞对于化疗药物阿霉素的敏感性,p63异构体的不同表达模式将有可能作为肝癌基因治疗的新靶点。

Effects of different patterns of p63 isoform expression on HCC growth induced by a cell cycle-specific toxin

Objective To study the effects of various patterns of p63 isoform expression on the Adriamycin sensitivity of hepatoma cells. Methods Hep3 Bcells with p53 deleted were studied.Hep3 Bcells in the logarithmic phase were treated with different concentrations of Adriamycin,i.e.0（Solvent control）,0.08,0.16,0.32,0.64,and 1.28μmol/L.An MTT assay was used to detect the survival rate of Hep3 Bcells.Flow cytometry was used to detect the rate of Hep3 Bcell apoptosis.A comet assay was used to evaluate DNA damage in Hep3 Bcells and Western blotting was used to detect levels of the proteins Tap63,DNp63,and Cytochrome c. Results Adriamycin significantly reduced the survival rate of Hep3Bcells;0.08μmol/L resulted in a survival rate of 89%,0.16μmol/L resulted in a survival rate of 71%,0.32μmol/L resulted in a survival rate of 54%,0.64μmol/L resulted in a survival rate of 38%,and 1.28μmol/L resulted in a survival rate of 22%.The rate of apoptosis of Hep3 Bcells was 16% with 0.08μmol/L of Adriamycin,28% with 0.16μmol/L of Adriamycin,42% with 0.32μmol/L of Adriamycin,56% with 0.64μmol/L of Adriamycin,and 78% with1.28μmol/L of Adriamycin.The extent of damage to the DNA of Hep3 Bcells was 2.9with 0.08μmol/L of Adriamycin,5.1with 0.16μmol/L of Adriamycin,7.2with 0.32μmol/L of Adriamycin,9.4with 0.64μmol/L of Adriamycin,and12.1with1.28μmol/L of Adriamycin.Levels of Tap63 and Cytochrome c protein expression were upregulated（P〈0.01）while there were no significant changes in the level of DNp63 protein expression.Thus,the ratio of Tap63/DNp63 increased significantly（P〈0.01）. Conclusion An increase in the Tap63/DNp63 ratio may significantly increase the sensitivity of Hep3 Bcells to Adriamycin.Various patterns of p63 isoform expression may be a new target for gene therapy to treat liver cancer.