探討A型流感病毒感染小鼠肺部幹細胞之特性

Characterization of Influenza A virus Infection in Mouse Pulmonary Stem/Progenitor Cells

趙苔伶(Tai-Ling Chao)

指導教授 ： 張淑媛

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00883

摘要

流感病毒藉由飛沫傳播，常引發急性且嚴重的呼吸道與肺部疾病，其中A型流感病毒曾引發多起世界性大流行造成上千萬人死亡，但仍不明瞭造成病人肺衰竭至死之原因。之前研究發現於肺部中存在一群帶有幹細胞標幟、表現Clara cell secretary protein (CCSP)且能分化成第一型或是第二型肺泡細胞的幹細胞，並發現此細胞可能是SARS-CoV感染人體的目標細胞，當此細胞受到感染後會失去原先修復肺組織的能力，進而造成肺組織嚴重損毀失去呼吸功能。而實驗室之前的研究發現A型流感病毒可感染幹細胞並造成細胞病變死亡，但不清楚其感染特性，因此本篇論文想要探究A型流感病毒感染幹細胞所具有的特性。

實驗首先利用實驗室病毒株PR8感染小鼠肺部幹細胞且觀察其於幹細胞中的生長趨勢，同時利用螢光染色發現幹細胞可表現α2,3及α2,6兩種唾液酸。接著為了探究病毒感染小鼠肺部幹細胞所表現的特色，以一般細胞株MDCK做為對照組，進行生長趨勢比較、病毒結合唾液酸試驗、病毒進入細胞試驗、病毒於細胞中的核酸複製能力以及比較新生成的病毒，發現MDCK細胞株生成的病毒量較高，而且在病毒結合唾液酸試驗及病毒進入細胞試驗上，病毒結合與進入幹細胞的量皆少於MDCK細胞株。在分析核酸複製能力之實驗中觀察到病毒NP基因的vRNA於幹細胞中生成速率較低。以蔗糖梯度超高速離心分析來自不同細胞新生成的病毒，發現來自幹細胞的病毒密度小於來自MDCK細胞株且以蛋白質電泳分析病毒組成，發現來自不同細胞的病毒其神經胺酸酶分子量大小不同。以帶有抗藥性重組病毒株感染幹細胞發現病毒生長趨勢不受抗藥性基因影響且具高度抗藥性。最後以H1N1臨床病毒株、新流感病毒株與H3N2臨床病毒株感染小鼠肺部幹細胞，但相較於實驗室病毒株PR8，臨床病毒株與新流感病毒株都無法有效的感染小鼠肺部幹細胞。

本論文研究當A型流感病毒感染小鼠肺部幹細胞時，僅有實驗室病毒株PR8可有效感染幹細胞，臨床病毒株與新流感病毒株則不易感染幹細胞。同時肺部幹細胞所製造的病毒量低於一般細胞株MDCK，其原因可能是病毒與唾液酸結合力、進入細胞能力及核酸複製能力皆較弱。此外，幹細胞可製造出密度較低的病毒顆粒，且當抗藥性病毒感染幹細胞時，生長趨勢未受影響且仍具有其抗藥性。

關鍵字

A型流感病毒 ； 小鼠肺部幹細胞

並列摘要

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.

並列關鍵字

Influenza A virus ； Mouse pulmonary stem/progenitor cells

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