出版年:

2001

研究生:

陳懿德

研究生(英文姓名):

Chen.I-Te

論文名稱:

感染腸病毒71型及單純疱疹病毒第一型所引發腦細胞基因表現的變化

英文論文名稱:

Profiling of gene expression patterns in neuroblastoma cell line SK-N-SH infected with enterovirus 71 and herpes simplex virus type

指導教授:

黎慶 ; 陳舜華

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46881026

學年度:

89

語文別:

中文

論文頁數:

86

關鍵詞:

腸病毒 ; 單純疱 ; 疹病毒 ; 生物晶片

英文關鍵詞:

EV71 ; HSV1 ; microarray ; bio-chip

被引用次數:

0

[ 摘要 ]

DNA microarray的技術提供了科學家們一個靈敏且快速的方法來了解細胞內部整體的基因表現，特別是可以幫助我們釐清在病原體侵入細胞時，支持或是抵禦病原體基因的表現。本論文首先利用microarray這項新的技術來找出腸病毒71型和單純疱疹病毒第一型感染神經細胞時的關鍵。經由這項技術，本研究分別找到了許多在腸病毒71型4643株或是單純疱疹病毒第一型kos株感染SK-N-SH神經細胞時發生變化的基因，其中包括egr-1、CD44、VDUP1、GST和PML等。這些基因表現的變化也被RT-PCR證實。在這些經過確認的基因之中，已知和細胞的生長和分化息息相關的基因 -- egr-1（early growth response protein 1），會因這兩種病毒的感染而有相似的上升趨勢，顯示出egr-1在病毒感染的調控路徑上可能扮演了相同的角色。所以我們進一步分析此基因受到腸病毒71型或單純疱疹病毒調控時的promoter活性。首先，這裡建構了不同刪減長度的egr-1 promoter到pGL3的載體：pEgr-1/-712代表含有全長之egr-1 promoter（full-length；從-712到+6），pEgr-1/-550、pEgr-1/-402、pEgr-1/-239和pEgr-1/-129分別代表含有從-550、-402、-239、-129到+6等不同刪減長度的egr-1 promoter。片段插在pGL3載體之冷光酶（luciferase）基因之前，然後大量複製這些載體並轉染（transfect）SK-N-SH細胞中，以偵測egr-1 promoter的活性。冷光分析的結果顯示在沒有感染的SK-N-SH細胞中，影響egr-1 promoter功能的主要區域是位於-402 ~ -550之間；腸病毒71型感染時主要的調控區域也是在此；單純疱疹病毒第一型感染時的主要調控區域則是在-550 ~ -712之間。此外，又利用antisense阻斷基因蛋白的合成，研究這些基因egr-1、CD44、V

[ 英文摘要 ]

DNA microarray technology allows scientists to address simple description about global gene expressions inside cells. In particular, it offers a sensitive and fast way to elucidate potential host genes whose expressions can assist or defend the invasion of a pathogen. The goal of the entire research project is to identify host genes that regulate enterovirus 71 (EV71) and herpes simplex virus type 1 (HSV-1) infections using cDNA microarray technology. Throughout these experiments, I have found that the expression of many genes, including egr-1, cd44, vdup1, GST and PML, changed during the infections of EV71 strain 4643 and/or HSV-1 strain kos in a neuroblastoma cell line SK-N-SH, suggesting that they are involved in the infection processes. The change of gene expressions was confirmed by RT-PCR. Among the identified genes, egr-1 (early growth response protein 1), whose transcriptional activity has been associated to proliferation and differentiation, was found to be induced similarly in SK-N-SH cells infected with EV71 or HSV-1, indicating that egr-1 plays a significant role in a common regulatory pathway for virus transmission. We therefore selected this gene for further characterization on the promoter activities regulated by EV71 or HSV-1. For this, I constructed the serial deleted egr-1 promoters, as follows: pEgr-1/-712 is the pGL3 luciferase-based plasmid containing the full-length egr-1 promoter (-712 to +6) cloned before the luciferase gene, whereas pEgr-1/-550, pEgr-1/-402, pEgr-1/-239, and pEgr-1/-129 were pGL3 inserted with the respective egr-1 promoter fragments from -550, -402, -239,or -129, to +6. These promoter constructs were then transfected into SK-N-SH cells to assay the activities of the egr-1 promoter regulated by viral infections. The result of the luciferase assay showed that the egr-1 promoter region between -402 ~ -550 as the most important region for basal expression of the protein in the cell line. The cis-regulatory element(s) i
n the egr-1 promoter that was responding to the induction by EV71 infection was located between -402~ -550, whereas the region regulated by HSV-1 was located further upstream of -550 ~ -712. In parallel, I investigated the physiological functions of the proteins including EGR-1, CD44, VDUP1, and P
ML-1, whose gene expressions were altered, in virus infections by using antisense technology. When some of these gene expressions were blocked, the virus proliferation would be limited. The results showed that the production of EV71 or HSV-1 was respectively decreased by he blockage of gene expression of cd44 or egr-1. My experiments have gained an insight into the molecular regulatory mechanism of virus infection mediated by these genes, which may further be used for research and development