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研究生： 洪士耘
Shih-yun Hung
論文名稱： 以微卡計量測細胞膜不同含量膽固醇和神經節苷脂之
Microcalorimetric and cytotoxic measurements of different cholesterol and ganglioside content plasma membrane PC12 cells exposed to various form of amyloid-beta
指導教授： 陳文逸
Wen-yih Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 98
語文別： 中文
論文頁數： 114
中文關鍵詞： 微卡計 、細胞膜 、細胞毒性 、神經節苷脂 、膽固醇 、類澱粉胜肽 、阿茲海默症
外文關鍵詞： plasma membrane, microcalorimetry, cytotoxic, ganglioside, cholesterol, amyloid-beta, Alzheimer''s disease
相關次數： 點閱：14  下載：0 
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    • 類澱粉蛋白(amyloid-?，A?)被認為是引起阿茲海默症的主因，當A?由單體在細胞膜上聚集成纖維狀結構時就會毒殺細胞，但是A?引起細胞毒性及導致疾病的機制目前還不清楚。近年研究指出，細胞膜上的神經節苷脂?GM1)和膽固醇的含量與A?的貼附和聚集有關。
    本研究以恆溫滴定微卡計作為即時熱流量之量測工具，測量細胞膜不同含量膽固醇和神經節苷脂之PC12細胞與不同聚集度之類澱粉蛋白作用後的反應熱，熱流數據計錄從細胞放入恆溫滴定微卡計到注射A?後並反應兩到三天。同時我們也在96孔盤內進行與微卡計內相同條件的實驗，透過這兩個實驗的結合期望能夠了解細胞在類澱粉蛋白的作用下其熱流量與細胞活性的關係。
    在96孔盤內的實驗結果顯示在48小時內，降低細胞膜GM1可抑制A?對細胞的毒性；另外降低細胞膜膽固醇也可抑制A?對細胞的毒性，而增加細胞膜膽固醇則會加強A?對細胞的毒性。
    將細胞培養在組織培養皿內並改變細胞膜上的膽固醇和GM1的含量後，取下培養皿上細胞放入微卡計內待平衡和細胞貼附後注射入不同聚集度的A??monomer rich, oligomer rich, fiber rich)，結果顯示熱流量與細胞活性及死亡率有高度相關，證明微卡計熱流量可做為細胞狀態的參考，而透過積分注射後之總放熱量並除以死亡細胞數，發現當系統內細胞毒性較強時，單位細胞死亡所放出的熱量會增加。

    ?-amyloid(A?) was believed to cause the prime factor of Alzheimer’s disease. The deposition and aggregation of A? on cell membrane led to cytotoxicity. However, the detail mechanism?was still unclear. Recent studies demonstrated that certan components of plasma membrane, ganglioside(GM1) and cholesterol play an influential role in the accumulation of A? on the plasma membranes.
    In this study, we applied isothermal titration calorimetry (ITC) as an “on-line” heat probe to identify the living cell systems. We changed the cholesterol and GM1 amount on PC12 cells plasma membrane in vitro and traced the thermodynamic activity of the interaction between A? and different contents plasma membrane PC12 cells by ITC. The heat flux data contained not only metabolic heat of cells but olso other reaction heat between A? and cells. Base on different experiment designs, different forms of A??(monomer rich, oligomer rich and fiber rich) were chosen. All heat flow is recorded by ITC from cell seeding to the end of experiment. We also redo the control experiment in 96 wells as a parallel experiment.
    Our results demonstrated that the reduction of cholesterol or GM1 contents on plasma membrane could diminish the A??cytotoxicity?in12, 24 and 48 hours but higher cholesterol content could cause higher cytotoxicity.
    The heat flow data from ITC was highly correlated to the MTT reduction and LDH release of parallel experiment. We demonstrated the heat flow from our ITC system could be a good indication of cell viability.
    By integrating the total heat dissipation after the injection of A?, the exothermic heat per dead cell enhanced when the system condition was more toxic to cell.

    摘要 Ⅰ Abstract Ⅱ 圖目錄 Ⅴ 表目錄 Ⅸ 第一章 緒論 1 第二章 文獻回顧 2 2.1 阿茲海默症（Alzheimer’s Disease，AD） 2 2.2 類澱粉胜肽（b-Amyloid） 7 2.2.1 類澱粉胜肽的聚集 10 2.3 PC-12細胞株 13 2.4 細胞膜上特定成份對類澱粉胜細胞毒性的影響 13 2.4.1 細胞膜上膽固醇（cholesterol）對類澱粉胜肽細胞毒性的影響 14 2.4.2 細胞膜上神經結苷酯（ganglioside，GM1）對類澱粉胜肽毒性的影響 16 2.4.3 細胞膜上膽固醇和神經結苷脂(GM1)與Ab聚集的機制 18 2.5 恆溫滴定微卡計（Isothermal Titration Calorimetry，ITC） 23 2.6以恆溫微卡計做為細胞放熱的偵測器 25 第三章 實驗藥品與儀器設備 30 3.1 實驗藥品 30 3.2 實驗設備 31 3.3 實驗方法 32 3.3.1 PC12細胞的培養 32 3.3.2 玻璃片覆蓋聚離胺酸(poly-D-lysine) 32 3.3.3 微卡計系統內的滅菌 33 3.3.4 A?的製備 33 3.3.5 改變細胞膜上膽固醇和GM1含量 37 3.3.5.1 降低細胞膜膽固醇含量 37 3.3.5.2 增加細胞膜膽固醇含量 37 3.3.5.3減少細胞膜GM1含量 37 3.3.6 細胞活性測試（MTT reduction）與細胞死亡測試（LDH release） 38 3.3.6.1 96孔盤內實驗 38 3.3.6.2 微卡計內細胞的測試 38 3.3.7 微卡計實驗流程 39 3.3.8 細胞死亡熱量之計算 40 第四章 結果與討論 41 4.1 不同濃度過氧化氫對PC12細胞的毒性及其熱流量 41 4.2 改變細胞膜組成後對細胞活性的影響以及培養在微卡計內細胞的活性 49 4.3不同聚合度Ab對降低GM1濃度細胞膜之PC12細胞和普通細胞的毒性 53 4.3.1 Fiber rich Ab對降低GM1濃度細胞膜之PC12細胞和普通細胞的毒性 53 及熱流量 53 4.3.2 Monomer rich Ab對降低GM1濃度細胞膜之PC12細胞和普通細胞的 59 毒性及熱流量 59 4.3.3 Oligomer rich Ab對降低GM1濃度細胞膜之PC12細胞和普通細胞的 65 毒性及熱流量 65 4.4 不同聚合度Ab對不同膽固醇含量細胞膜之PC12細胞的毒性及熱流量 71 4.4.1 Fiber rich Ab對不同膽固醇含量細胞膜之PC12細胞的細胞毒性及熱 71 流量 71 4.4.2 Monomer rich Ab對不同膽固醇含量細胞膜之PC12細胞的細胞毒性 77 及其熱流量 77 4.4.3 oligomer rich Ab對不同膽固醇含量細胞膜之PC12細胞的細胞毒性及 82 其熱流量 82 4.5 細胞死亡的放熱 87 4.5.1過氧化氫實驗 87 4.5.2 改變細胞膜GM1含量與不同結構Ab反應實驗 87 4.5.3 改變細胞膜膽固醇含量與不同結構Ab反應實驗 88 第五章 結論 89 第六章 參考文獻 90

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