吳益群臺灣大學：分子與細胞生物學研究所葉凱嘉Yeh, Kai-ChiaKai-ChiaYeh2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49893計畫性細胞死亡在多細胞生物體的發育過程中扮演著重要角色，它負責調控細胞數目的 琠w性以及避免因細胞死亡所引發的發炎反應。如果計畫性細胞死亡的機制出現缺陷將有可能引發自體免疫疾病。在計畫性細胞死亡的末期，死細胞的屍體必須由吞噬細胞清除乾淨，目前已知在線蟲裡有兩條具有相同功能的細胞吞噬路徑。一者為經由ced-2, ced-5,以及ced-12這條路徑；另一為經由ced-1, ced-6以及ced-7這路徑來達成細胞吞噬的目的。而在ced-2這條路徑裡，接受來自死亡細胞訊息的受器尚未完全找出。之前以RNAi的方式對線蟲進行一連串細胞吞噬缺陷突變株的screen，找出了一個tyrosine kinase： MER-1。在這篇報告中，我們試著釐清這個屬於tyro3 RTK家族成員的蛋白質MER-1在細胞吞噬路徑中所扮演的角色。由mer-1 在線蟲中胚胎進行RNA干擾的數據來看，胚胎的細胞屍體數目在2-fold時期前相較於野生種有顯著增加的趨勢，顯示出mer-1能影響細胞吞噬路徑的正常作用。而經由mer-1和ced-1, 6, 7, 2, 5, 10和12的雙重突變株cell corpse assay的結果來看，mer-1應是屬於ced-2, 5 和12的這條訊息傳遞路徑。MER-1具有一tyorsine kinase domain， 而根據kinase assay的結果證明， MER-1的tyrosine kinase domain在活體外確實具有酵素活性。Programmed cell death (PCD) plays an important role in the developmental process of multicellular organisms. After cells undergo PCD, cell corpses are recognized and phagocytosed by engulfing cells. There are two redundant genetic pathways for cell corpse engulfment in C. elegans. One is through ced-2, ced-5, and ced-12 pathway, and the other is ced-1, ced-6 and ced-7. In the former pathway, an engulfing cell receives the “eat-me” signal from apoptotic cells through phosphatidylserine receptor-1(PSR-1). In previous RNAi based screen for receptor tyrosine kinase that functions in cell corpse engulfment, mer-1 was identified. In this report showed that mer-1 RNAi increased cell corpses number in C. elegans’ embryo, especially at the 2-fold stage. This result indicates mer-1 may function in cell corpse engulfment pathway. Double mutant cell corpse analysis showed that mer-1 acts with ced-2, ced-5, ced-10 and ced-12 to control cell corpse engulfment. MER-1 has a potential tyrosine kinase domain. On the basis of kinase assay, MER-1 has autophosphorylation activity in vitro.中文摘要……………………………………………………………………………………...4 英文摘要……………………………………………………………………………………...5 前言 計畫性細胞死亡的概觀………………………………………………………………...6 線蟲模式動物的優點…………………………………………………………………...7 細胞屍體的吞噬作用…………………………………………………………………..10 線蟲的細胞屍體吞噬機制…………………………………………………………….12 MER tyrosine kinase受體蛋白參與在細胞屍體吞噬作用…………………………16 材料與方法 結果 MER-1預測是一個跨膜蛋白並包含一個具保守性的tyrosine kinase domain…………..23 mer-1轉錄產物(transcript) 5’端具有SL1 squence………………………………………...23 mer-1 的缺失會影響到胚胎發育早期細胞屍體吞噬的正常功能…………………………24 mer-1和psr-1很可能位於同一訊息傳遞路徑……………………………………………...25 mer-1很可能參與在ced-2,ced-5,ced-10和ced-12訊息傳遞路徑…………………………25 MER-1 KD的過量表現會有顯性抑制(dominant negative)的情形……………………….26 MER-1可能作用在吞噬細胞來影響吞噬作用的進行………………………….………….26 mer-1(tm632)突變株未有吞噬功能缺失的表型特徵…………………….…………………27 純化的MER-1蛋白具有autophosphorylation kinase activity……………………………28 純化的rabbit α-MER-1(e) antibody應可辨識到線蟲原生性的MER-1…………………28 討論 mer-1在細胞屍體吞噬訊息傳遞路徑中的定位…………………………………………….29 MER-1 kinase 的分子作用機制…………………………………………………………….30 Rabbit α-MER-1(e) antibody 很有可能辨識到線蟲原生性的MER-1蛋白……………..31 參考文獻………………………………………………………………………………………32 圖表1773818 bytesapplication/pdfen-US線蟲細胞吞噬計畫性細胞死亡C. eleganscell corpse engulfmentprogrammed cell deathotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49893/1/ntu-94-R91225017-1.pdf