【作者】 何琼琼；

【导师】 陈主初； 程瑞雪；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 丙型肝炎病毒(HCV)感染是目前影响人类健康的严重问题,它的持续感染可引起慢性肝炎、肝硬化和肝细胞癌(HCC)的发生,但对于HCV 持续感染导致慢性肝疾病的机理还不清楚。HCV 是一种RNA 病毒,无逆转录酶活性,只在细胞胞浆内复制,不能与宿主基因整合,主要是通过其表达的蛋白质起作用。尽管对于HCV 是否直接参与肝细胞恶性转化这一问题尚不清楚,但已有研究显示HCV 的结构蛋白C 和非结构蛋白NS3、NS4B、NS5A 具有致癌作用。其中HCV NS3 蛋白(核苷酸3420~5312,含631 个氨基酸残基)是一个多功能的蛋白质,其N 端1/3 具有丝氨酸蛋白酶活性,C 端2/3 则表现为解旋酶和NTP 酶活性,HCV NS3 介导的蛋白酶解过程是病毒在体内复制所必需。因HCV NS3 在病毒生活周期中起重要作用,并可和宿主蛋白相互作用,故其在HCV 相关性HCC 中的作用已成为研究的热点之一。HCV 为嗜肝细胞病毒,其自然宿主细胞是人或黑猩猩的肝细胞,采用非肝细胞作为研究体系,不能真实反映HCV 感染的自然进程,而以往的大部分研究均未采用肝细胞。因此本研究首次选用人源性永生化非瘤性肝细胞株QSG7701 作为研究对象,将HCV NS3 基因导入QSG7701 细胞,观察转染肝细胞生物学特性的改变。实验表明HCV NS3 转染的肝细胞(pRcHCNS3/QSG)显示出增殖旺盛的的特点,G0/G1 期细胞数目减少而S 期细胞数目增加。pRcHCNS3/QSG 细胞的倍增时间较空白质粒转染细胞更多还原

【Abstract】 Hepatitis C virus (HCV) infection is one of major worldwide health problem. Persistent infection with it can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). It is not well understood the mechanism of persistent viral infection and the pathogenesis of HCC. HCV, as a RNA virus, only replicates in the cytosol and fails to integrate with the host genome to activate oncogene. Most researches indicate that the interaction between HCV protein and liver cell gene/protein has been involved in the pathogenesis of HCC. Some evidences support that C, NS3, NS4B and NS5A have oncogenic potential,although it remains unclear if HCV exerts direct effects on liver cell transformation. HCV NS3 protein (nucleotide 3420 to 5312 with 631 amino acid residues) is a multi-functional viral protein. In addition to serine proteinase activity, which is located in the one-third of NS3 protein at the N-terminal end, helicase and nucleotide triphosphatase activities are identified in the C-terminal half of NS3 protein. The proteolytic events mediated by NS3 serine protease are believed to be essential for viral replication in vivo. HCV NS3 that plays a key role in the life cycle of virus and interacts with host cellular protein has become one of hotspots in recent research. As a hepacivirus, HCV natural host is the hepatocyte of human or chimpanzee. But the cell lines used by most researches were not based on HCV natural host cell─hepatocyte. In order to simulate the HCV infection model more clearly, non-tumorigenic human liver cell line QSG7701 was choosed and transfected with HCV NS3 plasmid. The biological behavior of transfected QSG7701 cells was observed. Flow cytometry analysis indicated that expression of HCV NS3 could accelerate the progression of G1 to S in cell cycle. Cell proliferation assay showed that the population doubling time in HCV NS3 transfected cells was much shorter than that in vector plasmid (pRcCMV) transfected cells and non-transfected cells (24h, 26h, 28h respectively). The cloning ratio of pRcHCNS3/QSG cells, pRcCMV/QSG cells and non-transfected QSG7701 cells was 33%, 1.46% and 1.11% respectively; the former is higher than the followed two. Tumor development was seen in nude mice inoculated with pRcHCNS3/QSG cells after 15 days. HE staining showed its hepatocarcinoma feature, and immunohistochemistry confirmed the expressions of HCV NS3 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/QSG cells andnon-transfected QSG7701 cells after 40 days. Our study showed for the first time that the HCV NS3 transformed QSG7701 cells and induced tumor development in human liver cells while the carcinogenesis mechanism of HCV NS3 is still unclear. Based on the advantages of proteomics on carcinogenesis studies, we turned to this new technology –“proteomics”which would help to comprehensively clarify the carcinogenesis in protein level and provide evidences for HCC diagnosis and treatment. According to this method, the total proteins from the pRcHCNS3/QSG cells and pRcCMv/QSG cells were separated by 2D electrophoresis respectively. A total of 1183±77 and 1035±82 spots were detected in pRcHCNS3/QSG cells and pRcCMv/QSG cells respectively. The IEF direction deviation of pRcHCNS3/QSG cells and pRcCMv/QSG cells is 0.978 ±0.136mm and 0.865 ±0.122mm respectively and the SDS-PAGE direction deviation of pRcHCNS3/QSG cells and pRcCMv/QSG cells is 0.978±0.136mm and 0.951±0.203 mm. Here the well-resolved and reproducible 2DE pattern of pRcHCNS3/QSG cells and pRcCMv/QSG cells was established. There were 920±60 spots matched between pRcHCNS3/QSG and pRcCMv/QSG cells. 55 spots were up regulated in pRcHCNS3/QSG cells and 40 spots up regulated in pRcCMv/QSG cells. Subsequently 21interesting protein spots were excised from gels and digested by trypsin in-gel respectively, then the matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was utilized to search the SWISS-PORT and TrEMBL database with Peptident software. We successfully identified 15 of those protein spots. Some proteins are involved in cell proliferation, apoptosis and immune response respectively. Much of them play an important role in cell signal transduction. The results suggested that hepatocyte transformation induced by HCV NS3 is a complex procedure. The balance between cell proliferation and apoptosis is broken in HCV NS3 transfected cells, which may lead cell to grow limitlessly and develop tumor. Further study with those differential expression proteins might provide a target for HCC diagnosis and treatment. More interestingly, signaling molecules such as Ras、P38 and HD53 were upregulated in transfected cells,which provided an evidence that the MAPK signal transduction pathway may play an important role in cell transformation. The MAPK molecule is activated by phosphorylation and inactivated by dephosphorylation. Because MALDI-TOF-MS analysis can not provide sequence information directly, it is essential to study the expression of MAPK in HCV NS3 transfected cells. Western blot analysis showed that pRcHCNS3/QSG cells resulted in a higheractivity of phosphorylated ERK1/2, P38 and JNK in comparision with pRcCMv/QSG cells and QSG7701 cells while the basal expression of ERK1/2 and P-38 showed no difference,which suggested that the MAPK signal molecules may play an important role in HCV NS3 related carcinogenesis. Immunoprecipitation analysis displayed higher lever of tyrosine phosphorylation in HCV NS3 transformed cells. Although the exact protein above-mentioned still need to be identified, we showed here that HCV NS3 may affect cell signaling pathway through activating tyrosine kinase and following with several cytoplasmic signaling molecules such as MAPKs, and result in hepatocyte transformation and tumor development.更多还原