The Receptors Mediated Hepatitis C Virus Invation Of Mouse Hepatoma Carcinoma Cell

Hepatitis C virus is an RNA virus and pathogen via blood transmission, with a global epidemic. About 170 million people are chronically infected all over the world, with about 3% infection rate. There are about 3.5 million new cases every year.After infected, it causes acute hepatitis, about which 75% above turns into chronic hepatitis, gradually severse develops fibrosis and liver cirrhosis even liver cancer. Due to the lack of appropriate vaccine and limited therapy, the prevention and treatment of HCV has been a worldwide problem. So many researchers have made attention to it. Because of HCV strict species-specificity, it can only infect human and chimpanzee. If they were used in the experimental study, there will be many problems, such as expensive, ethics. The development of research about HCV has been hampered by the lack of available cell and animal models.HCV invading target host cells is a complex process, involving several cellular factors. At present, the majority of people believe that there are five the main receptors in HCV cell-entry, including low density lipoprotein receptor(LDLR), tetraspanin CD81, scavenger receptor class B type?I(SR-BI), tight junction proteins claudin-1(CLDN1)and occludin(OCLN). Moreover CD81 and OCLN are the minimum two molecules. HCV combined with low density lipoprotein(LDL) or very low density lipoprotein(VLDL) to form lipoviral particles(LVPs). Firstly glycosaminoglycans(GAGs) and LDLR gathered LVPs to the surface of liver cell membrane. After the other HCV receptors worked together, clathrin-mediated and low p H caused membrane fusion. Finally HCV neucleocapsid released into the cytoplasm.Only can CD81 and OCLN cause the virus to enter target cells. Whether the weight or combination of several other receptors can further promote HCV virus entry, that remains to be thinking. And in-depth researches about the function of receptors in HCV early life cycle are lack. Therefore, the role of related receptors should be further discussed, which mediate HCV early entry into hepatocytes participate in the virus infection.The purpose of this study is to build transgenic mouse cell model, which could be infected with HCV in vitro. Further study about the function of HCV receptors will be made on the basis of these cell models. The results of the study are as follows:1. Screen transgenic mouse hepatoma carcinoma cell strains. We developed transgenic cell strains by lentivirus transfection, which can express HCV multiple receptors stably. In order to package lentivirus, recombinant lentivirus vectors expressing HCV receptors and packing plasmids were transferred into cells by liposome, which vectors had been built and saved in our lab; cell line Hepa1-6 were incubated with lentivirus particles;transgenic cells were obtained by fluorescence activated cell sorting and antibiotics. Many metheds were used to detect purpose moleculars expressing in transgenic cells, such as RT-PCR, laser confocal microscopy and western blot.2. Transgenic mouse hepatoma carcinoma cell strains can make HCVpp entry. Transgenic cells were incubated with HCVpp virus, which expressed HCV envelope protein E1 and E2. Virus entry were decided by the detection of luciferase expressed in cells. The resultes showed that HCV virus could get into transgenic mouse cell strains SCCO/Hepa1-6 and LSCCO/Hepa1-6, which expressed four or five receptors. And tandem expression of receptor molecules had no influence on virus infection.3. Transgenic cell strains SCCO/Hepa1-6 and LSCCO/Hepa1-6 can cause HCV virus natural infection, and receptors sequences of LDLR-SR-BI-CD81 and CLDN-1-OCLN are more likely to promote virus invade target cells. Through direct infection of HCV positive serum, we confirmed that the transgenic cell strains could make virus natural entry, and LDLR could cause more serum virus entry; another transgenic cell strain LCSCO/Hepa1-6 was developed by lentivirus transfection, which expressed HCV receptors in the series of LDLR-CD81-SR-BI and CLDN-1-OCLN; to discuss effect on virus invasion in different receptor molecules orders, we used HCV positive serum to infect transgenic cells LSCCO/Hepa1-6 and LCSCO/Hepa1-6; By detecting HCV RNA copies in the transgenic cells and fluorescence expression of virus protein, we found that LSCCO/Hepa1-6 cells were more likely to cause HCV entry; in other words, the order of receptors expressed on the surface of LSCCO/Hepa1-6 cells was more conducive to virus invasion.4. Due to different virus form, HCVcc infection was different from virus natural infection and the role of LDLR was not obvious in HCVcc virus entry. By comparing HCVcc virus binding and entry between SCCO/Hepa1-6 and LSCCO/Hepa1-6, we found that LDLR was little impact on HCVcc early infection; To elimiante the effect of molecules in series and evacuate the influence of tandem expression, we reformed the saved lentivirus vectors and constructed other vectors, which made molecules single express and more close to the body of the state; Hepa1-6 cells were transfected with new recombinant vectors by liposome and incubated with HCVcc; by detecting HCV RNA copies in cells, we found that virus entry were basically same when the receptor molecules expressed as tandem or alone; Considering different virus form and receptors in nature infection and HCVcc infection, we compared the role of LDLR and SR-BI in the process of HCVcc into target cells and found that the SR-BI was an essential molecular in HCVcc infection. These results showed that HCVcc infection and natural infection were different and LDLR mediating HCV positive serum into target cells had little influence on HCVcc infection.Summarizing all the data, this study constructed transgenic mouse hepatoma carcinoma cell strains, including SCCO/Hepa1-6, LSCCO/Hepa1-6 and LCSCO/Hepa1-6, which stably expressed different quantity and series sequence of human HCV receptors. We confirmed that these transgenic cells could make virus entry and be used to research HCV as cell models in virto; By comparing cell strains LSCCO/Hepa1-6 with LCSCO/Hepa1-6, we proved that HCV virus particles interacted priority with SR-BI when they entered the host cells; On the basis of cell strains SCCO/Hepa1-6 and LSCCO/Hepa1-6, we confirmed that SR-BI was a capital molecule and the role of LDLR was not obvious in HCVcc early entry stage; These cell models built on this research provided the platform for screening or evaluation of early HCV anti-infection drugs, and even development of vaccine. The function of HCV related receptors in its early life cycle were made some further studies on the foot of cell models; at the same time, we also recognized the importance of the study about HCV natural infection; these results also provided new trains of thread for the future research about HCV anti-infection drug targets.