流感病毒感染不同物種肺部幹細胞之機制及其鑑定

在先前的經驗中，我們成功的在無血清（serum-free）的環境下培養出小鼠肺部上皮細胞。其中有一群細胞對SARS冠狀病毒有高度敏感性，且同時表現Oct-4、 SSEA-1、Sca-1及CCSP等幹細胞/肺部前驅細胞標記。這些細胞是否會被其他呼吸道病毒所感染目前還不清楚。在本研究中，我們發現這些細胞同樣能夠被H3N2、 H5N1等流感病毒感染。在細胞被H3N2病毒感染後，在肺部前驅細胞中的HMGB1蛋白會從細胞核移動到細胞質，並釋放出來，同時也偵測到發炎前驅細胞激素（pro-inflammatory cytokines）IL-1β, IL-6及TNF-α的表現增加。然而，我們也發現小鼠肺部上皮細胞並不會被 H5N2所感染。因此，我們嘗試去尋找更適合的細胞來做病毒研究。到目前為止，狗的腎細胞（MDCK）是最常使用的細胞，但為了能夠研究病毒感染時，宿主最真實的反應，我們利用同樣方法培養出雞的肺部上皮細胞。這些細胞能夠被H5N1、H5N2、H6N1和H7N7等病毒感染，且在培養過程中，觀察到有細胞群落（colony）的形成。進一步用PCR及Q-PCR來鑑定這些細胞的特性，我們發現這些細胞表現了鹼性磷酸酶（alkaline phosphatase）、PouV（chicken Oct-4 homologue）、SOX2等幹細胞的標記。同時細胞也表現E-cadherin、pulmonary surfactant protein等肺部上皮細胞的標記。因此，我們認為雞的肺部上皮細胞可以作為很好的病毒研究平台。另一方面，我們利用pRSV-TEX質體（含有SV40 T抗原）轉染入細胞，建立雞的肺部上皮細胞株。在繼代培養6代後，發現細胞與初代培養的細胞表現同樣的特性，且帶有SV40 T抗原。若持續維持及繼代有機會成為雞的肺部上皮細胞株。

關鍵字

流感病毒；肺臟；幹細胞；細胞激素

並列摘要

We have reported a serum-free primary culture system to generate pulmonary epithelium stem/progenitor cells (Oct-4+/SSEA-1+/Sca-1+ and CCSP+) with surrounding stroma cells in mouse. These stem/progenitor cells were the target cells for SARS-CoV infection in primary cultures. However, the targeting potential of these cells for other lung infection-associated viruses remains unclear. We demonstrated the susceptible ability of these colony cells for influenza virus infection, such as H3N2 and H5N1.After H3N2 infection, HMGB1(High mobility group box 1 ) predominantly translocated to the cytoplasm in colony cells, and the pro-inflammatory cytokines like IL-1β, IL-6 TNF-α were up-regulated. However, H5N2 virus could not infect the cells at all. So far, the available cell line to study influenza virus infection was MDCK, which derived from kidney cells of dog. To examine that the pulmonary epithelium stem/progenitor cells were the first target for virus infection, and study the cross-species infection of influenza virus, we established both chicken and mouse pulmonary stem/progenitor cells and to compare their susceptibility of virus infection. The chicken lung epithelial cells were susceptible for H5N1, H5N2, H6N1 and H7N7 virus. Further experiments by RT-PCR and Q-PCR showed the expression of stem cell markers in the chicken lung cells, including alkaline phosphatase (cAP), cPouV, cSOX2, but not cNanog. The E-cadherin, pulmonary surfactant protein, and telomerase were also demonstrated to be expressed in cells. These observation suggested that the chicken pulmonary stem/progenitor cells are able to serve as a good cell model to study the influenza virus infection. Furthermore, we transfected the chicken pulmonary epithelial cells with plasmid pRSV-TEX to establish a cell line suitable for cell-based vaccine production. These cells were subcultured to passage 6, and expressed the same marker as primary cells. SV40 large T antigen also can be detected in passage 6 cells.

並列關鍵字

influenza virus ； lung ； stem cell ； cytokine

參考文獻

Bardiya, N., and Bae, J.H. (2005). Influenza vaccines: recent advances in production technologies. Appl Microbiol Biotechnol 67, 299-305.

Beigel, J., and Bray, M. (2008). Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res 78, 91-102.

Bermejo, J.F., and Munoz-Fernandez, M.A. (2004). Severe acute respiratory syndrome, a pathological immune response to the new coronavirus--implications for understanding of pathogenesis, therapy, design of vaccines, and epidemiology. Viral Immunol 17, 535-544.

Bernard, G.R. (2005). Acute respiratory distress syndrome: a historical perspective. Am J Respir Crit Care Med 172, 798-806.

Bryan, T.M., and Reddel, R.R. (1994). SV40-induced immortalization of human cells. Crit Rev Oncog 5, 331-357.

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流感病毒感染不同物種肺部幹細胞之機制及其鑑定

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