论文部分内容阅读
目的端粒酶线粒体转位与原发性肝细胞癌化疗耐药的关系。方法采用RNA干扰技术下调肝细胞Shp-2表达;靶向定位线粒体mito-hTERT慢病毒载体上调肝细胞线粒体hTERT表达;Western blot和激光共聚焦技术检测端粒酶hTERT线粒体转位;免疫荧光技术测定caspase-3活化状态;采用WST-8实验检测肝癌细胞耐药状态;Mito-Sox探针染色检测线粒体活性氧(ROS);Q-PCR检测线粒体DNA(mtDNA)氧化损伤频率。结果 (1)增强外源性线粒TERT表达后,肝癌细胞对于CDDP、5-FU、DOX耐受性增强,并且能减少药物应激导致的凋亡激活过程;(2)抑制Shp-2表达导致CDDP刺激下HepG2-ShShp-2细胞线粒体TERT表达增加;(3)HepG2-ShShp-2细胞对CDDP和5-FU的耐药性增加,RI分别为2.094和1.863;(4)HepG2-ShShp-2细胞线粒体ROS生成减少、mtDNA氧化损伤水平降低。结论线粒体hTERT转位可能导致肝癌细胞对化疗药物的耐药性增加;线粒体hTERT通过降低ROS生成和mtDNA氧化损伤水平,保护线粒体功能并减少线粒体途径的凋亡激活,参与肿瘤细胞对化疗药物产生耐药。
Objective To investigate the relationship between telomerase translocation and chemoresistance in primary hepatocellular carcinoma. Methods The expression of Shp-2 in hepatocytes was down-regulated by RNA interference. Mitochondrial mitochondrial hTERT expression was up-regulated by targeting mitochondrial mito-hTERT lentiviral vector. Mitochondrial translocation of telomerase hTERT was detected by Western blot and confocal laser scanning microscopy. Caspase-3 activation state; WST-8 assay was used to detect the drug resistance of hepatocellular carcinoma cells; Mito-Sox probe was used to detect mitochondrial reactive oxygen species (ROS); Q-PCR was used to detect the oxidative damage of mitochondrial DNA. Results (1) Enhanced expression of exogenous mitochondria TERT enhanced hepatocarcinoma cells to CDDP, 5-FU, DOX enhanced, and can reduce the activation of apoptosis induced by drug stress; (2) Inhibition of Shp-2 expression (3) The resistance of HepG2-ShShp-2 cells to CDDP and 5-FU increased, RI were 2.094 and 1.863 respectively; (4) HepG2-ShShp-2 cells increased the mitochondrial TERT expression in HepG2-ShShp- 2 cells reduce the generation of mitochondrial ROS, mtDNA oxidative damage decreased. Conclusion Mitochondrial hTERT translocation may lead to increased resistance of chemotherapeutic drugs to hepatocellular carcinoma cells. Mitochondrial hTERT protects mitochondria from activation of mitochondrial function by decreasing ROS production and mtDNA oxidative damage, medicine.