2014-03-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649171摘要：H7N9 病毒感染人類的致病機轉仍未完全被了解，而現行 in vitro 流感病毒研究所使用的犬腎細胞（MDCK）與猴腎細胞，皆無法代表病毒在人類細胞上的感染模式。 人類呼吸道上皮細胞培養（primary human airway epithelium cell culture）是最常被用於模擬呼吸道上皮環境的系統。這種培養基採取內外兩種不同介面（air-liquid interface），讓呼吸道上皮細胞暴露在空氣中，而分化為偽纖毛細胞，杯狀細胞等，並有正常的生理活動包括纖毛運動，分泌黏液等。研究顯示，人類呼吸道上皮細胞培養系統，接近真實 in vivo 人類呼吸道細胞對感染或其他刺激的反應。 除瞭解病毒感染誘發的宿主防禦機制，在 H7N9 流感疫苗發展過程中，也將藉由人類呼吸道上皮細胞及其他傳統細胞株，做為疫苗接種後血清中和試驗的平台。此平台也可作為減毒疫苗的初步安全性預測，優於其他非人類的細胞株培養系統。 台大醫院小兒感染症實驗室已能建立人類呼吸道上皮細胞培養系統。針對 H7N9 禽流感病毒，將藉此平台，期達成下列目標：(1)確認不同的流感病毒（H1N1, H3N2, H7N9）能在人類呼吸道上皮細胞培養繁殖。(2)了解不同的流感病毒在人類呼吸道上皮細胞的受體差異（alpha 2,3 或 alpha 2,6）。(3)感染不同流感病毒後，人類呼吸道上皮細胞所釋放的細胞激素差異。(4) 感染不同流感病毒後，人類呼吸道上皮細胞所造成的氧化傷害。(5)利用人類呼吸道上皮細胞，進行疫苗試驗後的血清中和實驗及安全性評估。 <br> Abstract: By the end of October 2013, the National Health and Family Planning Commission of China had reported 139 cases with 45 fatalities infected by a new human H7N9 influenza A virus. This virus is a reassortant of hemagglutinin (HA) subtype 7 (H7), neuraminidase (NA) subtype 9 (N9) gene, and several other gene segments from different avian IAVs. It also contains mutations that facilitate infection in mammals, which could pose a pandemic threat. Analysis of gene sequences indicates that the new H7N9 virus is better adapted to infect humans than other avian influenza A viruses. However, the pathogenesis and the mechanism for human cellular adaption of H7N9 infection are still not fully understood. Current in vitro cell culture systems that are permissive for influenza virus replication include MDCK from canine and Vero from monkey, which are not representative for host infection in humans. Human airway epithelium (HAE) cultured in an “air-liquid interphase” is a commonly used model to study the biology of human airways. HAE cells are obtained by biopsy, brushing, surgery, lung transplant or post-mortem and cultured on a collagen substratum in the presence of growth factors, with the apical surface of the cells exposed to air. HAE can differentiate and form a pseudostratified epithelium that expresses tight junctions, develops cilia, and produces mucin. Previous reports have shown that the well-differentiated HAE cell culture is a good representation of in vivo airway epithelial transcriptome. Vaccines are the key defense elements against pandemics. HAE cells that preserve the cellular properties of human airway epithelium can be a surrogate of human airway for serum neutralization tests in preclinical trials of influenza vaccine. Moreover, the HAE cell system can be applied to the screening of restricted replication of live attenuated influenza vaccine candidates. A HAE cell culture system has been established in the Pediatric Virology Lab of National Taiwan University Hospital (NTUH) since 2011. We believe the HAE system is useful to study the pathogenesis, and the transcriptional profiles of human cellular responses to the H7N9 infection. Therefore, in this proposal we will access: a) the replication abilities of seasonal H1N1, H3N2, and human H7N9 to HAE cells; b) the binding affinities of the HA/NA protein from the three viruses to different sialic acid receptors (alpha 2,3 and alpha 2,6) of HAE cells; c) cytokine released from apical and basilar aspects of HAE cells after viral infections; d) cellular oxidative stress response of HAE cells after viral infections; e) serum neutralization for vaccine development, and restricted replication of live attenuated influenza vaccine candidates.人類呼吸道上皮細胞培養H7N9禽流感致病機轉血清中和human airway epitheliumH7N9avian influenzapathogenesisserum neutralization test.