| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 99 === Influenza viruses spread by droplets, often causing acute and severe respiratory and lung diseases, including influenza A virus most often caused by a worldwide pandemic. Found in previous studies one stem cells with stem cell markers Oct-4+, expressed clara cell secretory protein (CCSP) and differentiated into type I or type II alveolar cells, and also found that stem cells could be infected with SARS-CoV as human target cells. SARS-CoV infected cells lose the ability to repair the injury tissue, thus causing serious damage to lung tissue. In our laboratory previous study, found that influenza viruses can infect stem cells and cause cytopathic effect. But the characteristics of virus infection in stem cells are still not clear. The purpose of the research is exploring the influenza virus infected stem cells with such characteristics.
First experiment use laboratory strain PR8 to infect mouse pulmonary stem/progenitor cells, then observe viral growth curve in cells. Then, we found that stem cells express both two kinds sialic acid, α2,3-linkage and α2,6-linkage, by using immunefluorescence assay. Second, in order to explore the performance characteristics of virus when infected stem cells, we tested viral growth trend, viral binding and entry abilities and viral genome replication efficiency in stem cell and control group MDCK cells, found that two types of cells infected with the same dose, MDCK cell lines available to reach higher viral load. In viral binding and entry assay, compare to MDCK cells, stem cells have less virus binding and entry. In viral genome replication assay, we observed that vRNA synthesis of viral NP gene was much slower in stem cells than MDCK cells. Third, using sucrose gradient ultra- centrifugation analyzes viral characteristics from different cells, and got the result of virus from stem cell has lower density and the molecular weight of its neuraminidase is also smaller. Then, to explore whether virus with resistance mutations resistant to Tamiflu or not, we made recombinant virus with drug-resistant NA gene and infected cells. In this experiment, we found resistance virus can grow as well as wild type virus, also can resist to Tamiflu in stem cells. Finally, stem cells can not be strongly infected by using two H1N1 clinical strains, one pandemic H1N1 strain and one H3N2 clinical strain.
This research found the possible reasons of why less viruses be made in mouse pulmonary stem/progenitor cells: lower binding and entry ability to stem cells and lower viral RNA replication efficiency. And also found virus particle from stem cells has lower density and smaller neuraminidase to MDCK cell lines. Finally, resistance NA gene not only did not disturb virus growth but also gave the ability to resist Tamiflu in stem cells.
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