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標題: | 利用雷射光分別誘導細胞膜與黑色素體之損傷 Triggering plasma membrane or melanosome damage by light |
作者: | Pei-Jou Liu 劉珮柔 |
指導教授: | 楊維元 |
關鍵字: | 細胞自噬,泛素,細胞膜,黑色素體,CALI, autophagy,ubiquitin,plasma membrane,melanosome,CALI, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 胞器的品質控制對於生物的生存非常重要,過去眾多的文獻揭露許多疾病的發生與胞器品質控制失衡有關。受損的胞器一般皆經由細胞自噬作用(autophagy)清除,細胞自噬是細胞內自我分解機制的一種,而在過去不同的研究當中,也都證實細胞自噬參與在胞器的品質控制中。
於此,我們使用光誘導策略對C2C12肌肉纖維母細胞(C2C12 myoblast cell)的細胞膜,以及MNT-1黑色素細胞(MNT-1 melanocytes)和B16F1黑色素細胞 (B16F1 melanocytes)的黑色素體(melanosome)進行破壞。利用光敏劑(photosensitizer)的吸光特性,使用雷射光激發光敏劑產生活性氧化物(ROS),進而誘導細胞自噬作用進行。在雷射共軛焦顯微鏡下運用此方法,我們可以控制在單一細胞中,欲產生損傷的位置與損傷程度。 在本篇研究中,我們觀察到,在細胞膜受損後,細胞會立即進行修復,修復的模式可以大致區分為兩類:胞吞作用(endocytosis)與脫除作用(shedding)。根據損傷程度的不同,細胞會傾向啟動不同的作用機制,在細胞膜受損嚴重時,脫除作用的發生比率會提高,而在較輕微的損傷狀況下,則以胞吞作用為主。但不論受損程度之強弱,在受損細胞膜附近都能觀察到細胞膜的內噬(internalization),並且會被泛素(ubiquitin)標記,進而進行細胞自噬而被降解。而在受損黑色素體的研究中,我們發現可見光的中間波段可以用來誘導黑色素體的損傷,並且這些受損黑色素體會被泛素標記。而這樣被泛素化的現象,表示受損的黑色素體有可能會藉由細胞自噬作用來清除。 概括而言,我們使用光敏劑搭配光誘導策略對細胞膜和黑色素體進行破壞,利用此方法,可以控制對單一細胞欲產生損傷的位置與損傷程度。在細胞膜受損時,細胞會因著損傷程度的不同而有不同的修復策略,然而不論在何種損傷程度下,部分的膜會被內噬並進一步被泛素標定而進行細胞自噬降解。在誘導黑色素體損傷的部分,我們提供了一個更加簡便與精準的方法,可以幫助我們更深入去探討黑色素體的品質控制。 Quality control of organelles is crucial for survival and numerous studies have revealed the relationship between organelles dysregulation and diseases. Damaged organelles are believed to be degraded through autophagy, and different research have proved the involvement of autophagy which is a “self-eating” degradative machinery in cell. Here, we applied the light-induced scheme to impair plasma membrane of C2C12 myoblast cells, and the melanosomes in MNT-1 and B16F1 melanocytes. With the photosensitizer-based methodology, the photosensitizers were bleached to induce autophagy in the selective region. By combining the scheme with confocal microscope, we can spatiotemporally control the position and the severity in a single cell. In our observation, after the plasma membranes injury, cell would immediately undergo membrane repair through endocytosis and shedding. Depending on the severity, cells tended to conduct shedding at the strong damaged condition and favored endocytosis at the weak damaged condition. No matter how severe the damage, the internalization of damaged membrane usually happened, and the internalized membranes were targeted by ubiquitin to execute autophagy. As for melanosome, the middle of the visible spectrum could properly trigger the damage with ubiquitin targeting. And the ubiquitin recruitment indicated that the damaged melanosome may process autophagy for degradation. In summary, we utilized the photosensitizer-based impairment to spatiotemporally induce damaged plasma membrane and melanosome. In our observation, cells can choose their plasma membrane quality control procedure through sensing the severity of the injuries, and autophagy participates in the elimination of internalized membranes. The induction of damaged melanosome provides a precise and convenient methodology to discover the mechanism of melanosome quality control. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69220 |
DOI: | 10.6342/NTU201801623 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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