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研究生:胡志煌
論文名稱:細胞激素IL-3與GM-CSF融合蛋白質結構及功能的探討
論文名稱(外文):Structural and Functional Studies of Cytokine IL-3 and GM-CSF Fusion Proteins
指導教授:許宗雄許宗雄引用關係
指導教授(外文):T.H. Hseu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:78
中文關鍵詞:細胞激素
外文關鍵詞:cytokine
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細胞激素幫助調節免疫作用細胞的發展,而且有些細胞激素本身也擁有直接作用的功能。如同荷爾蒙作為內分泌系統的傳遞物質一般,細胞激素扮演著免疫系統傳遞角色。但是它們不像內分泌的荷爾蒙有傳遞到遠處的影響,通常只是區域性的作用。
根據過去的臨床研究顯示,IL-3和GM-CSF在基因治療上是最具潛力的兩個細胞激素,此外,由於IL-3和GM-CSF具有共同的生化活性,結構上有許多相同的特徵,又具有極相似的αβ受器,且兩者都是表現在被活化的T細胞。因此,我們架構了兩個基因:IL-3─GM-CSF及GM-CSF─IL-3,經由DNA transfection到PA317細胞,使用37.5 μg/ml的G418來選取含有IL-3─GM-CSF及GM-CSF─IL-3基因的群落;並培養2至3天後,大量的融合蛋白質(IL-3─GM-CSF及GM-CSF─IL-3)就釋放到培養液中,收取其上清液,利用ELISA可得到這兩個融合蛋白質所含細胞激素IL-3及GM-CSF的濃度,再藉由免疫沉澱法及西方點墨法來測其蛋白質的分子量;最後我們利用TF-1細胞來檢測兩個融合蛋白質(IL-3─GM-CSF及GM-CSF─IL-3),與細胞激素IL-3和GM-CSF對TF-1細胞的生長差異。
結果顯示,我們得到的兩個融合蛋白質(IL-3─GM-CSF及GM-CSF─IL-3),的確具有細胞激素IL-3及GM-CSF。此外,在生化上的測試,也證明了IL-3─GM-CSF及GM-CSF─IL-3融合蛋白質具有加成的作用。最後,由 ” Insight II ” 程式的模擬,得到IL-3─GM-CSF及GM-CSF─IL-3融合蛋白質最可能的結構。
Cytokines assist in regulating the development of immune effector cells, and some cytokines possess direct effector functions of their own. Just as hormones serve as messengers of the endocrine system, so cytokines serve as messengers of the immune system. Unlike endocrine hormones, though, which exert their effects over large distances, cytokines generally act locally.
Clinical studies suggested that cytokines IL-3 and GM-CSF might have significant potential in gene therapy. Additionally IL-3 and GM-CSF share several common biological activities and structure features. Theirαβ receptors are almost alike and are both expressed in activated T cells. Consequently, we have constructed two genes one is IL-3─GM-CSF, the other is GM-CSF─IL-3. By way of DNA transfection, the PA137 cell used G418 to select the gene that we had transfectd. We pick up the clones and cultured them for 2 to 3 days. the fusion protein was released to the medium and then harvested the culture medium. Using ELSIA, we can detect the concentration of IL-3 and GM-CSF from two fusion protein (IL-3─GM-CSF and GM-CSF─L-3). To determine the molecular weigh of those two fusion protein, we used immunoprecipitation and wertern blotting. Finally, we check the growth curve by culture TF-1 cell in two fusion protein and cytokine (IL-3 and GM-CSF).
The result indicated that we get the IL-3─GM-CSF and GM-CSF─IL-3 fuion protein consisted of cytokines IL-3 and GM-CSF. With only a few exceptions, in biological activity test, we have suggested that IL-3─GM-CSF and GM-CSF─IL-3 fuion protein really have a double effect. By predicting the structure of two fusion protein, we demonstrated a highly consistent structure of IL-3─GM-CSF and GM-CSF─IL-3 fusion protein by using “Insight II” program.
目 錄
第一章 緒論……………………………………………………………1
一、 細胞激素之簡介……………………………………………1
二、 IL-3的簡介…………………………………………………3
三、 GM-CSF的簡介……………………………………………5
四、 IL-3與GM-CSF在臨床上的應用…………………………7
五、 實驗目的……………………………………………………8
第二章 實驗材料與方法………………………………………………9
一、常用試劑………………………………………………………9
二、實驗方法………………………………………………………14
(一)Gene construct…………………………………………14
(二)細胞培養…………………………………………………16
(a)基礎培養液之配備……………………………………16
(b)細胞繼代培養…………………………………………17
b-1附著型細胞…………………………………………17
b-2懸浮型細胞…………………………………………18
(c)DNA transfection………………………………………18
(d)DNA合成分析(Thymidine incorporation assay) ……19
(e)生長曲線(growth curve)之測試……………………20
(三) 酵素免疫分析(ELISA) ………………………………21
(四)免疫沉殿法(immunoprecipitation) ……………………23
(五)西方點墨法(western blotting) ……………………………25
(a)SDS PAGE…………………………………………25
(b)蛋白質之轉印………………………………………26
(c)免疫點墨法(immunoblot) …………………………27
第三章 實驗結果……………………………………………………29
一、 IL-3―GM-CSF及GM-CSF―IL-3基因的架構………29
二、 IL-3―GM-CSF及GM-CSF―IL-3融合蛋白質細胞
激的表現……………………………………………………29
三、 IL-3―GM-CSF及GM-CSF―IL-3融合蛋白質分析……30
四、 生長曲線(growth curve)之測試……………………………31
五、 DNA合成分析(Thymidine incorporation assay) …………31
六、 IL-3―GM-CSF及GM-CSF―IL-3融合蛋白質結構模擬…32
第四章 討論………………………………………………………34
第五章 參考文獻………………………………………………………40
第六章 圖表……………………………………………………………51
附錄…………………………………………………………………62

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