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研究生:盧正祐
研究生(外文):Cheng-You
論文名稱:弓蟲感染星狀膠細胞誘發基質金屬蛋白酶-12增加及彈性蛋白降解
論文名稱(外文):Matrix metalloproteinase-12 increase and elastin degradation in astroglia infected with Toxoplasma gondii
指導教授:蔡崇弘蔡崇弘引用關係賴世展
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:60
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弓蟲(Toxoplasma gondii)是一種專性細胞內寄生的原蟲,此蟲能感染絕大部分的哺乳類動物,包括人。當弓蟲感染宿主後常誘發弓蟲性腦炎(toxoplasmic encephalitis)。本研究我們利用弓蟲速殖子(TS-4 strain)感染星狀膠細胞(astroglia)的細胞模式進行研究,探討基質金屬蛋白酶-12(matrix metalloproteinase-12, MMP-12)和其受質彈性蛋白(elastin)的變化之致病機轉。結果顯示在感染組細胞培養液中發現MMP-12蛋白表現量在感染後第24小時和第48小時開始有明顯的增加。同樣的,彈性蛋白表現量在相同時間點也有顯著的增加。抑制組的星狀膠細胞於感染後分別加入0.01mM、0.1mM、0.5mM和1mM等四種不同濃度的MMPs專一酵素抑制劑GM6001共同培養。結果顯示0.5mM及1mM 等兩種濃度,細胞崩解的程度及MMP-12和彈性蛋白的蛋白表現量相較於感染組也有明顯的降低。試驗結果顯示在弓蟲感染星狀膠細胞的發展過程中,MMP-12扮演降解彈性蛋白的角色。

Toxoplasma gondii is an obligate intracellular protozoa in the mammal animals, including human. The host infected with T. gondii wich can induce toxoplasmic encephalitis. In this study, astroglia infected with TS-4 strain T. gondii tachyzoite and to investigate the changes of matrix metalloproteinase-12 (MMP-12) and its substrate elastin in the pathogenesis. The results showed that MMP-12 was found in the cell clutered medium of T. gondii infected-astroglia and significantly increased at 24hrs and 48 hrs post-infection (PI). Similarly, the expression of elastin was also increased at the same time points. The inhibitory test of astroglia infected with T. gondii was co-cultured with four different concentrations of MMPs inhibitor GM6001 (0.01mM, 0.1mM, 0.5mM and 1mM). The concentrations of GM6001 on 0.5mM and 1mM were significantly reduced the astroglia degradation, and the expression of MMP-12 and elastin in astroglia infected with T. gondii. These results suggested that MMP-12 may contribute to elastin degradation occurring in astroglia infected with T. gondii.

中文摘要 ………………………………………………………………I
英文摘要 ………………………………………………………………II
目錄 ……………………………………………………………………1
壹、背景及動機 ………………………………………………………4
一、弓蟲(Toxoplasma gondii)………………………………4
二、星狀膠細胞(astroglia)…………………………………5
三、基質金屬蛋白酶(matrix metalloproteinases,MMPs)…7
四、彈性蛋白(elastin)………………………………………9
五、MMPs專一性的酵素抑制劑GM6001 …………………………10
六、研究動機 ……………………………………………………11
貳、材料方法 …………………………………………………………12
一、細胞株 ………………………………………………………12
二、弓蟲速殖子之製備 …………………………………………12
三、體外試驗:弓蟲速殖子感染星狀膠細胞 …………………12
四、弓蟲速殖子感染細胞後之細胞培養液的收集 ……………13
五、基質金屬蛋白酶抑制試驗…………………………………13
六、西方點墨法(Western blotting) ………………………14
七、免疫共同沉澱(Co-Immunoprecitation) ………………15
八、統計分析 ……………………………………………………16
九、試驗設計 ……………………………………………………16
参、實驗結果 …………………………………………………………17
一、觀察弓蟲速殖子感染星狀膠細胞後之形態變化 …………17
二、細胞培養液中MMP-12蛋白表現量 …………………………17
三、細胞培養液中彈性蛋白蛋白表現量 ………………………17
四、酵素抑制劑GM6001對弓蟲速殖子感染星狀
膠細胞之抑制作用 …………………………………………18
五、加入酵素抑制劑GM6001對細胞培養液中MMP-12
蛋白表現量的影響 …………………………………………19
六、加入酵素抑制劑GM6001對細胞培養液中彈性蛋白
蛋白表現量的影響 …………………………………………19
七、MMP-12與彈性蛋白之相互作用 ……………………………20
肆、討論 ………………………………………………………………21
伍、參考文獻 …………………………………………………………27
陸、圖、表
圖一、觀察弓蟲速殖子感染星狀膠細胞之形態變化 ………………47
圖二、弓蟲速殖子感染星狀膠細胞之細胞培養液中基質金屬
蛋白酶-12(matrix metalloproteinases-12, MMP-12)
蛋白質的表現 …………………………………………………48
圖三、弓蟲速殖子感染星狀膠細胞之細胞培養液中
彈性蛋白(elastin)蛋白質的表現…………………………49
圖四、觀察MMPs專一酵素抑制劑GM6001對弓蟲速殖子
感染星狀膠細胞之抑制作用 …………………………………50
圖五、弓蟲速殖子感染星狀膠細胞並加入DMSO和MMPs
專一酵素抑制劑GM6001,在感染第24小時
對細胞培養液中基質金屬蛋白酶-12(matrix
metalloproteinases-12, MMP-12)蛋白量表現的影響……51
圖六、弓蟲速殖子感染星狀膠細胞並加入DMSO和MMPs
專一酵素抑制劑GM6001,在感染第24小時對
細胞培養液中彈性蛋白(elastin)蛋白量表現的影響……52
圖七、基質金屬蛋白酶-12(matrix metalloproteinases-12,
MMP-12)與彈性蛋白(elastin)免疫共同沉澱試驗………54
附錄 ……………………………………………………………………55


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