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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林泰元(Thai-Yen Ling) | |
| dc.contributor.author | Yu-Ming Lin | en |
| dc.contributor.author | 林佑名 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:42:46Z | - |
| dc.date.available | 2013-09-11 | |
| dc.date.copyright | 2008-09-11 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-31 | |
| dc.identifier.citation | Bardiya, N., and Bae, J.H. (2005). Influenza vaccines: recent advances in production technologies. Appl Microbiol Biotechnol 67, 299-305.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40214 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:42:46Z (GMT). No. of bitstreams: 1 ntu-97-R95443017-1.pdf: 2867877 bytes, checksum: be3ea3aaa238055adf0937226b88610e (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 縮寫表...................................................i
中文摘要................................................ii ABSTRACT...............................................iii 第一章 緒論..............................................1 1.1 A型流行性感冒病毒...................................2 1.1.1 A型流行性感冒之背景及流行現況.....................2 1.1.2 A型流行性感冒病毒之結構及基因體...................2 1.1.3 A型流行性感冒病毒之感染途徑及機制.................3 1.1.4 A型流行性感冒病毒感染所造成之宿主反應.............4 1.1.5 A型流行性感冒的治療與預防.........................5 1.1.6 流感疫苗的發展....................................6 1.2 研究動機............................................7 第二章 材料與實驗方法....................................8 2.1 實驗材料與藥品.......................................9 2.2 細胞株培養..........................................11 2.3 肺部上皮細胞的初級培養（primary culture）...........11 2.4 病毒培養、力價測定及感染............................13 2.5 鹼性磷酸酶（alkaline phosphatase）活性測定..........14 2.6 細胞免疫染色（Immunocytochemistry）.................14 2.7 細胞上sialic acid-α2,3-galactose及sialic acid-α2,6-galactose的偵測.........................................15 2.8 反轉錄聚合酶連鎖反應................................15 2.9 同步定量聚合酶連鎖反應（Real-time PCR）.............18 2.10 雞肺部上皮細胞株建立...............................20 第三章 實驗結果.........................................22 3.1 H3N2病毒感染小鼠肺部上皮細胞的情形..................24 3.2小鼠肺部上皮細胞sialic acid-α2,3-galactose及sialic acid-α2,6-galactose的分佈情形...............................24 3.3小鼠肺部上皮細胞受到H3N2病毒感染後發炎前驅細胞激素（proinflammatory cytokine）的表現......................24 3.4小鼠肺部上皮細胞受到H3N2病毒感染後HMGB1的表現........25 3.5 H5N1及H5N2病毒感染小鼠肺部上皮細胞的情形............25 3.6 雞肺部上皮細胞群落（colony）型態與形成..............26 3.7 H5N1及H5N2病毒感染雞肺部上皮細胞的情形..............26 3.8 H6N1及H7N7病毒感染雞肺部上皮細胞的情形..............26 3.9雞肺部上皮細胞sialic acid-α2,3-galactose及sialic acid-α2,6-galactose的分佈情形...............................27 3.10雞肺部上皮細胞特性之鑑定............................27 3.11雞肺部上皮細胞在不同培養天數下與幹細胞有關之mRNA表現......................................................27 3.12雞肺部上皮細胞株的建立..............................28 3.13 pRSV-TEX轉染並繼代培養之雞肺部上皮細胞之鑑定.......28 第四章 討論.............................................29 第五章 結論與未來展望...................................36 參考文獻................................................65 | |
| dc.language.iso | zh-TW | |
| dc.subject | 流感病毒 | zh_TW |
| dc.subject | 肺臟 | zh_TW |
| dc.subject | 幹細胞 | zh_TW |
| dc.subject | 細胞激素 | zh_TW |
| dc.subject | stem cell | en |
| dc.subject | cytokine | en |
| dc.subject | lung | en |
| dc.subject | influenza virus | en |
| dc.title | 流感病毒感染不同物種肺部幹細胞之機制及其鑑定 | zh_TW |
| dc.title | Identification of the Pulmonary Stem/Progenitor Cells From Different Species as the Target Cells for Influenza Virus Infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 郭明德(Ming-Der Kuo),鄭明珠(Ming-Chu Cheng),黃彥華(Yen-Hua Huang) | |
| dc.subject.keyword | 流感病毒,肺臟,幹細胞,細胞激素, | zh_TW |
| dc.subject.keyword | influenza virus,lung,stem cell,cytokine, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| 顯示於系所單位： | 藥理學科所 | |
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