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研究生:李嘉凌
研究生(外文):Chia-Lin Lee
論文名稱:藉由晚期短暫性UL94啟動子調控的巨細胞病毒pp71蛋白所造成的影響
論文名稱(外文):The effect of cytomegalovirus pp71 driven by the late temporal UL94 promoter
指導教授:龔思豪
指導教授(外文):Szu-Hao Kung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:人類巨細胞病毒殼膜區間蛋白
外文關鍵詞:HCMVtegument protein
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人類巨細胞病毒的感染為最主要造成免疫功能不全病人的高罹病率及高死亡率,特別是器官移殖及愛滋病病患。雖然抗人類巨細胞病毒的化學治療藥劑已經被發展建立;然而,長期地給予藥物治療,伴隨著治療的效果,會促使抗藥性病毒株的產生。因此,我們欲建立一基因修飾的細胞株,希望可以用來監測人類巨細胞病毒對抗病毒藥的敏感性。
人類巨細胞病毒的pp71蛋白(由UL82基因所表現的殼膜區間蛋白)已被證實可增加子代病毒的產量,其可能的機制為它能轉激活迅早期啟動子,而增加病毒的裂解循環。雖然pp71蛋白在病毒複製晚期大量表現,但是此表現卻一直被忽略。因此建立一穩定細胞株,命名為U373MG-UL94p-FlagUL82-IRES-EGFP,使得pp71蛋白及綠色螢光蛋白在人類巨細胞病毒感染下能在病毒複製晚期表現。此細胞株是以質體穩定轉染U373MG細胞株來建立的;而質體上含有人類巨細胞病毒真晚期UL94啟動子,來控制下游UL82基因及綠色螢光蛋白基因的表現。另外,以另一穩定細胞株U373MG-UL94p-SEAP-IG作為控制組,此細胞株亦由UL94啟動子控制綠色螢光蛋白的表現,但不表現外生性pp71蛋白的表現。
此穩定細胞株U373MG-UL94p-FlagUL82-IRES-EGFP對於CMV感染的感受度與傳統的MRC5雙套細胞相似。而且此細胞株在以病毒感染後一天即可偵測到綠色螢光蛋白的表現,但控制組細胞株則需至感染後三天才可明顯地偵測到綠色螢光蛋白。隨著感染時間的增加,感染後六至八天U373MG-UL94p-FlagUL82-IRES-EGFP細胞株可觀察到綠色螢光細胞群聚,而在控制組細胞株上則看不到綠色螢光群聚。當處以抗巨細胞病毒藥物(Ganciclovir)時,對藥物敏感的病毒,則不會有綠色螢光群聚的產生;而對藥物具有抗藥性的病毒株則依舊可觀察到綠色螢光蛋白的產生。穩定表現針對於pp71轉錄序列的小干擾核糖核酸的細胞株能夠明顯地減少病毒感染後所產生的綠色螢光的數目及強度。也顯示表現pp71蛋白的UL82基因可以作為一個新穎的抗病毒作用的標的。
因此,U373MG-UL94p-FlagUL82-IRES-EGFP穩定細胞株或許可以運用來針對人類巨細胞病毒做快速診斷、偵測病毒對藥物的感受度及評估針對pp71蛋白所設計的藥物。
Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality among immunocompromised patients, especially recipients of transplants and patients with acquired immunodeficiency syndrome (AIDS). Anti-CMV chemotherapeutic agents have been developed; however, prolonged administration of the drugs has prompted the emergence of drug-resistant CMV strains, compromising the efficacy of the therapy. Therefore, we propose to develop a genetically modified cell line to hopefully monitor sensitivity to anti-CMV drugs.
An HCMV tegument protein (encoded by the UL82 gene), has been shown to enhance the yield of progeny virus. The underlying mechanism has partially been its ability to transactivation of the immediate early (IE) promoter, resulting in the increased level of the virus lytic cycle. However, the role that the pp71 plays in the late phase of the viral cycle is largely elusive although the expression of pp71 is upregulated at this stage. Thus a stable cell line, designated U373MG-UL94p-FlagUL82-IRES-EGFP, was established to permit the pp71 and the green fluorescent protein (GFP) to be expressed late in the replication cycle of CMV. The cell line was developed by stable transfection of U373MG cell line with a plasmid encoding the UL82 and the GFP genes driven by a CMV true late promoter, the UL94 promoter. A stable line that only allows GFP expression under the control of the UL94 promoter (devoid of expression of ectopic pp71) was also established as a negative control.
The susceptibility of the U373MG-UL94p-FlagUL82-IRES-EGFP stable line to CMV infection appears to be similar to that of the MRC5 diploid cells. The U373MG-UL94p-FlagUL82-IRES-EGFP cell line exhibited detectable GFP as early as one day postinfection, while the control cells did not display GFP signal until 3 days postinfection. As the infection proceeds, GFP foci were seen on U373MG-UL94p-FlagUL82-IRES-EGFP cells 6-8 days postinfection whereas GFP foci were not seen on the infected control cells. In the presence of anti-CMV drug (ganciclovir), the GFP foci were not seen with the U373MG-UL94p-FlagUL82-IRES-EGFP cells infected with drug-sensitive CMV strain while the GFP foci were intact when infected by drug-resistant isolates. Stable cell lines expressing the short interfering RNA targeting pp71 transcript significantly reduced the number and intensity of GFP (+) cells of the infected U373MG-UL94p-FlagUL82-IRES-EGFP cells, indicating that the pp71 would serve as an alternative molecular target for anti-CMV agents.
The U373MG-UL94p-FlagUL82-IRES-EGFP cells could be used for rapid diagnosis, determination of antiviral susceptibility, and assessment of potential compounds against pp71 protein.
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