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| 題名: | 人體內臟幼蟲移行症之病原犬蛔蟲誘發Jurkat T lymphocytes進行凋亡之分子機制 |
| 作者: | 周嘉玫 |
| 貢獻者: | 醫學科學研究所 |
| 日期: | 2010 |
| 上傳時間: | 2010-10-20 12:02:03 (UTC+8) |
| 摘要: | 犬蛔蟲是人畜共通寄生蟲病常見的感染原之一，人類可因誤食犬蛔蟲受孕蟲卵而感染而造成犬蛔蟲症。在犬蛔蟲的小鼠模式裡，目前發現犬蛔蟲幼蟲能在受感染並啟動免疫反應的動物宿主體內長時間存活，然而卻無法解釋犬蛔蟲幼蟲逃避宿主的免疫撲殺的機轉為何。
本論文對人體內臟幼蟲移行症之病原犬蛔蟲是否可藉排泌性抗原誘發免疫細胞進行凋亡以逃避宿主的免疫補殺；或發炎細胞是否存在抗凋亡的機制進而導致慢性發炎的可能原因進行探討。
利用不同濃度(5、15、25、50、100 μg/ml)的犬蛔蟲幼蟲排泌性抗原或不同數量的犬蛔蟲幼蟲(150隻、300隻)與human Jurkat T lymphocyte cell line (clone E6-1)進行共同培養12、24、48或72小時後，發現以25 μg/ml 犬蛔蟲幼蟲排泌性抗原與human Jurkat T lymphocyte共同培養24小時後有最佳的致細胞凋亡的效果，然而隨著抗原量及共同培養時間的增加，細胞抗凋亡蛋白分子Bcl-2及Bcl-xL亦有明顯增加的趨勢。此外以300隻的犬蛔蟲幼蟲與human Jurkat T lymphocyte共同培養72小時後有明顯的凋亡表現，因此時cleaved caspase-8有顯著表現。
綜合上述結果推論犬蛔蟲幼蟲入侵宿主後可藉由幼蟲所分泌的排泌性抗原及蟲體本身引致human Jurkat T lymphocyte進行凋亡以逃避宿主的免疫撲殺。
Toxocara canis is one of the most commonly reported zoonotic helminth infections in the world. Humans can get toxocariasis by ingesting infective embryonated eggs. In current studies of T. canis infections using the murine model, researchers found that T. canis larvae could live in paratentic host such as mice for a long time. However, there’s no evidence to prove that T. canis larvae can evade the killing reaction controlled by host immunity.
Here, we intended to investigate whether human visceral larva migrans pathogen Toxocara canis may trigger apoptosis in inflammatory cells by releasing excretory-secretory (TcES) antigens that benefit larval evasion of immunological attacks, or whether there exists anti-apoptosis mechanisms in inflammatory cells that lead to chronic inflammation.
We use different concentrations (5、15、25、50、100 μg/ml) of TcES or different amount (150 or 300) of T. canis larvae co-cultured with human Jurkat T lymphocytes cell line (clone E6-1) for 12~72 hrs to explore the underline mechanism of larval evasion in host as assessed by flow cytometry and western blotting approaches. Our results indicated that 25 μg/ml TcES posed the strongest apoptotic effects on human Jurkat T lymphocyte for 24 hrs co-culture; whereas the expression of anti-apoptotic proteins of Bcl-2 and Bcl-xL increased with the increased concentration of TcES and co-culture time as well. In addition, 300 T. canis larvae are able to trigger apparently apoptosis of human Jurkat T lymphocyte for co-culture of 72 hrs due to enhanced caspase-8 expression.
Based on above findings, we postulate that T. canis larvae may evade immunological attacks by releasing excretory-secretory materials or larvae per se through triggering human Jurkat T lymphocyte apoptosis. |
| 關聯: | 54頁 |
| 描述: | 縮寫表 ……………………………………………………………… P1
中文摘要 …………………………………………………………… P2
英文摘要 ………………………………………………………… P3~P4
壹、緒論
一、犬蛔蟲(Toxocara canis)的生活史 ………………………… P5
二、人體犬蛔蟲症 ……………………………………………… P6~P7
三、人體犬蛔蟲症之流行病學 ………………………………… P7~P8
四、犬蛔蟲症臨床診斷與治療 ………………………………… P8~P9
五、犬蛔蟲症之實驗動物模式研究與T-lymphocytes於犬蛔蟲症之角色 … P9~P11
六、細胞凋亡 ………………………………………………… P11~P12
貳、研究動機 ………………………………………………… P13~P14
参、研究目的與實驗設計 ………………………………………… P15
肆、實驗方法與步驟
一、犬蛔蟲成蟲採集與感染型蟲卵之取得 ……………………… P16
二、犬蛔蟲感染性幼蟲孵育與排泌性抗原之取得 ………… P16~P17
三、細胞株之取得與培養 ………………………………………… P17
四、犬蛔蟲幼蟲排泌性抗原與Human Jurkat T lymphocytes共同培養 ……… P17
五、犬蛔蟲幼蟲與Human Jurkat T lymphocytes共同培養 …P17~P18
六、 利用Flow cytometry analysis偵測細胞凋亡或抗凋亡的變化 …………… P18
七、利用western blotting檢測細胞凋亡相關的蛋白分子的表現 ………… P18~P19
八、結果統計分析 ………………………………………………… P19
伍、結果
一、犬蛔蟲蟲卵收集與幼蟲孵育 ………………………………… P20
二、以western blotting確認所收集的培養液是否含有犬蛔蟲幼蟲排泌性抗原(TcES) …………………………………………………… P20
三、Flow cytometry偵測TcES與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 ……………………………… P20~P22
四、western blotting偵測TcES與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 …………………………… P22~P23
五、利用western blotting偵測T. canis larvae與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 ………… P23~P24
陸、討論 ……………………………………………………… P25~P28
柒、參考文獻 ………………………………………………… P29~P40
Figures 1~7 ……………………………………………………P41~P50
Appendix 1~4 ………………………………………………… P51~P54
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