CAJ | 학술논문

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。
목적：단립매선립체전위여원발성간세포암화료내약적관계。방법채용RNA간우기술하조간세포Shp-2표체；파향정위선립체mito-hTERT만병독재체상조간세포선립체hTERT표체；Western blot화격광공취초기술검측단립매hTERT선립체전위；면역형광기술측정caspase-3활화상태；채용WST-8실험검측간암세포내약상태；Mito-Sox 탐침염색검측선립체활성양（ROS）；Q-PCR 검측선립체 DNA （mtDNA）양화손상빈솔。결과（1）증강외원성선립TERT표체후，간암세포대우CDDP、5-FU、DOX내수성증강，병차능감소약물응격도치적조망격활과정；（2）억제 Shp-2표체도치 CDDP 자격하HepG2-ShShp-2세포선립체TERT표체증가；（3）HepG2-ShShp-2세포대CDDP화5-FU적내약성증가， RI분별위2.094화1.863；（4）HepG2-ShShp-2세포선립체ROS생성감소、mtDNA양화손상수평강저。결론선립체hTERT전위가능도치간암세포대화료약물적내약성증가；선립체hTERT통과강저ROS생성화mtDNA양화손상수평，보호선립체공능병감소선립체도경적조망격활，삼여종류세포대화료약물산생내약。
Objective This study focused on the role of mitochondrial translocation of hTERT in multidrug resistance of HCC. Methods HepG2, HepG2-Negative (HepG2 transfected with negative plasmid) and HepG2-ShShp-2 (HepG2 transfected with Shp-2 ShRNA plasmid) were used. Sensitivity of the cells to chemotherapeutic drugs was assessed by WST assays. The distribution of hTert within mitochondria was detected by Western blot and immunofluorescence combined with laser scanning confocal microscopy. The mitochondria ROS generation in cells was demonstrated by flow cytometry. The level of damaged mtDNA was illustrated by real-time PCR. Results Exogenous mitochondrial TERT overexpression enhanced insensitivity to CDDP, 5-FU, DOX and reduced apoptosis activation under drug stress. HepG2-ShShp-2 showed increasing hTERT translocated from the nuclei to the mitochondria after CDDP treated. HepG2-ShShp-2 showed higher resistance to CDDP, 5-FU and DOX. The RI of the three drμgs was 2.094, 1.863 and 1.066 in contrast to HepG2 control groups. HepG2-ShShp-2 indicated the elevation in mitochondria ROS generation while the reduction of damaged mtDNA under the chemotherapeutic stress. Conclusions In total, tumor cells may become resistant to chemotherapeutic stress throμgh hTERT-mediated mitochondrial protection. Mitochondria translocated hTERT exerts protective effect on mitochondrial function throμgh decreasing mtDNA damage, and reducing ROS generation.