He Qiong-Qiong, Cheng Rui-Xue, Sun Yi, Feng De-Yun, Chen Zhu-Chu, Zheng Hui
Department of Pathology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan Province, China.
World J Gastroenterol. 2003 Mar;9(3):474-8. doi: 10.3748/wjg.v9.i3.474.
To study the effect of hepatitis C virus nonstructural protein 3 c-terminal deleted protein (HCV NS3-5') on hepatocyte transformation and tumor development.
QSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 c-terminal deleted protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological behavior of transfected cells was observed through cell proliferation assay, anchorage-independent growth and tumor development in nude mice. The expression of HCV NS3 and c-myc proteins in the induced tumor was evaluated by immunohistochemistry.
HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. Cell proliferation assay showed that the population doubling time in pRcHCNS3-5' transfected cells was much shorter than that in pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning ratio of cells transfected with pRcHCNS3-5', pRcCMV and non-transfected cells was 33 %, 1.46 %, 1.11 %, respectively, the former one was higher than that in the rest two groups (P<0.01). Tumor development was seen in nude mice inoculated with pRcHCNS3-5' transfected cells after 15 days. HE staining showed its feature of hepatocarcinoma, and immunohistochemistry confirmed the expressions of HCV NS3 and c-myc proteins in tumor tissue. The positive control group inoculated with HepG2 also showed tumor development, while no tumor developed in the nude mice injected with pRcCMV and non-transfected cells after 40 days.
1.HCV NS3 c-terminal deleted protein has transforming and oncogenic potential. 2. Human liver cell line QSG7701 may be used as a good model to study HCV NS3 pathogenesis.
研究丙型肝炎病毒非结构蛋白3羧基末端缺失蛋白（HCV NS3-5'）对肝细胞转化及肿瘤发生发展的影响。
采用脂质体介导将质粒pRcHCNS3-5'（表达HCV NS3羧基末端缺失蛋白）转染QSG7701细胞，并用G418筛选。分别用PCR和免疫组织化学方法检测HCV NS3基因及蛋白表达。通过细胞增殖实验、非锚定依赖性生长实验及裸鼠体内肿瘤发生发展实验观察转染细胞的生物学行为。用免疫组织化学方法评估诱导肿瘤中HCV NS3及c-myc蛋白表达。
转染质粒pRcHCNS3-5'的QSG7701细胞中HCV NS3高表达，阳性信号位于细胞质。细胞增殖实验显示，pRcHCNS3-5'转染细胞群体倍增时间明显短于pRcCMV转染细胞及未转染细胞（分别为24小时、26小时、28小时）。pRcHCNS3-5'转染细胞、pRcCMV转染细胞及未转染细胞的克隆率分别为33%、1.46%、1.11%，前者高于后两组（P<0.01）。接种pRcHCNS3-5'转染细胞的裸鼠15天后出现肿瘤生长。HE染色显示其具有肝癌特征，免疫组织化学证实肿瘤组织中有HCV NS3及c-myc蛋白表达。接种HepG2的阳性对照组裸鼠也出现肿瘤生长，而接种pRcCMV转染细胞及未转染细胞的裸鼠40天后未出现肿瘤生长。
1.HCV NS3羧基末端缺失蛋白具有转化及致癌潜能。2.人肝细胞系QSG7701可作为研究HCV NS3致病机制的良好模型。
