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研究生:蔡易達
研究生(外文):Tsai, Yida
論文名稱:利用人類多瘤性病毒,JC病毒,殼體作為人類膀胱癌基因治療載體之研究
論文名稱(外文):Investigation Of Developing a Gene Delivery Vector Using The Human JC Virus-like Particle To Inhibit Human Urinary Bladder Carcinoma Growth
指導教授:張德卿
指導教授(外文):Chang, Deching
口試委員:江明格王梅林沈正煌
口試委員(外文):Chiang, MingkoWang, MeilinShen, Chenghuang
口試日期:2012-07-19
學位類別:碩士
校院名稱:國立中正大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:51
中文關鍵詞:人類多瘤性病毒JC病毒膀胱癌
外文關鍵詞:JC virus-like particleurinary bladder carcinoma
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人類多瘤性病毒,JC病毒 (JCV),可感染免疫功能低下患者的腦部神經膠細胞,而造成進行性多病灶腦白質病變。JCV會潛伏在人類的腎臟及泌尿道,包含腎盂、輸尿管及膀胱。這意味著JCV可能感染膀胱。結構蛋白VP1是JCV主要的殼體蛋白,可以自行組裝成類病毒殼體。此類病毒殼體可以包裹外源性的DNA並送入人類細胞進行表達。在這次研究裡,表達綠螢光蛋白的報告基因 (GFP) 和會產生單純疱疹病毒胸苷激酶 (HSV-TK) 的自殺基因分別於大腸桿菌體內被類病毒殼體包裝,形成gfp-VLP和tk-VLP,在細胞實驗時,可感染人類膀胱癌細胞TSGH-8301和HT-1197,並表達蛋白質。類病毒殼體在裸鼠動物模型下,可專一性的感染人類膀胱癌腫瘤。經由腹腔注射Acyclovir到已處理過tk-VLP的裸鼠,可發現人類膀胱癌腫瘤的生長明顯受到抑制。結果顯示未來有可能利用JCV的類病毒殼體作為基因輸送載體到人類膀胱腫瘤進行治療。
The JC virus (JCV) infects human oligodendrocytes and consequently causes progressive multifocal leukoencephalopathy (PML) in patients with immune deficiency. JCV latently persists in kidney and urinary tract (renal pelvis, ureter and urinary bladder) indicating that JCV may infect human urinary bladder. The recombinant major structure protein, VP1, of JCV can self-assemble to form a virus-like particle (VLP). It has been shown that the VLP can package and deliver exogenous DNA into human cells for gene expression. In this study, a reporter gene, green fluorescence protein (gfp), and a suicide gene, the herpes simplex virus thymidine kinase (tk), were encapsidated into VLP in E. coli. The gfp-VLP and tk-VLP were used to specifically target human bladder cancer cell lines (TSGH 8301 and HT-1197) in cell culture. The VLP was used to specifically target human bladder carcinoma cells (HT-1197) in a nude mouse model. Intraperitoneal injection of acyclovir (ACV) in the tk-VLP treated mice greatly reduced tumor volume of bladder cancer cells. The findings suggest that it is possible to develop a gene delivery vector by using the JCV VLP to treat human urinary bladder carcinoma in the future.
中文摘要 1
Abstract 2
一、 背景介紹 5
1. 膀胱癌的介紹 5
2. 膀胱癌的基因治療 8
3. 人類多瘤性病毒,JC病毒 10
二、 材料與方法 13
1. 細胞株及細胞培養 13
2. 純化gfp-VLP與tk-VLP 14
3. 血液凝集試驗 (Hemagglutination assay,HA) 15
4. 蛋白質電泳 (Electrophoresis) 16
5. 西方墨點法 (Western blot) 16
6. Virus-like particles內含之核酸萃取 17
7. 聚合酶連鎖反應 (Polymerase Chain Reaction,PCR) 17
8. 利用轉染 (Transfection) 送入pEGFP-N3 18
9. 利用gfp-VLP假感染 (Pseudoinfection) 18
10. 以tk-VLP配合Acyclovir (ACV) 毒殺膀胱癌細胞株 19
11. 細胞存活率分析 (Cell viability test) 19
12. 裸鼠及裸鼠飼養 20
13. 以gfp-VLP感染裸鼠皮下之膀胱癌腫瘤 21
14. 以tk-VLP配合ACV抑制裸鼠皮下膀胱癌腫瘤生長 21
三、 結果 22
1. 製備gfp-VLP或tk-VLP 22
2. 利用gfp-VLP感染膀胱癌細胞株 23
3. 以tk-VLP配合Acyclovir (ACV) 毒殺膀胱癌細胞株 24
4. 以gfp-VLP感染裸鼠皮下之膀胱癌腫瘤 25
5. 以tk-VLP配合ACV抑制裸鼠皮下膀胱癌腫瘤生長 26
四、 討論 27
五、 參考文獻 31
六、 實驗結果圖 40
圖1、以血液凝集測試破菌後的上清液內含的VLP titer。 40
圖2、以血液凝集測試經蔗糖濃度梯度純化後的gfp-VLP titer。 41
圖3、以血液凝集測試經蔗糖濃度梯度純化後的tk-VLP titer。 42
圖4、以SDS-PAGE分析經蔗糖濃度梯度純化後的VLP。 43
圖5、濃縮後的VLP,分析其純度及內含核酸。 44
圖6、以gfp-VLP感染TSGH-8301膀胱癌細胞株。 45
圖7、共軛焦顯微鏡觀察gfp-VLP感染HT-1197膀胱癌細胞株。 46
圖8、以trypan blue exclusion分析tk-VLP對膀胱癌細胞之毒殺作用。 47
圖9、以trypan blue exclusion分析tk-VLP對HT-1197之毒殺作用。 48
圖10、共軛焦顯微鏡觀察VLP攜帶之GFP於HT-1197腫瘤之表現。 49
圖11、VLP攜帶TK基因,抑制HT-1197腫瘤之生長。 50
圖12、VLP攜帶TK基因,抑制HT-1197腫瘤生長之量化圖。 51


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