出版年:

2000

研究生:

溫又曄

研究生(英文姓名):

Wen Yu-Ye

論文名稱:

腸病毒71型感染四株哺乳類細胞株之比較

英文論文名稱:

Comparative studies on EV71 infection in four mammalian cell lines

指導教授:

劉校生老師

指導教授(英文姓名):

Liu Hsiao-Sheng

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:

s46874079

學年度:

88

語文別:

中文

論文頁數:

0

關鍵詞:

腸病毒71型

被引用次數:

0

[ 摘要 ]

摘要
感染腸病毒71型的臨床病徵包括：手足口病、皰疹性咽峽炎、無菌性腦膜炎、類似小兒麻痺病徵的肢體麻痺及腦炎等。雖然臨床病徵顯示腸病毒71型可能具有嗜神經細胞特性，但目前為止，其真正的致病機制及細胞喜好性仍不清楚。本研究的目的為藉由觀察腸病毒71型感染四株哺乳類細胞株：Vero（Africa green monkey kidney），SK-N-SH (human neuroblastoma)，U373MG (human glioblastoma) 及 HEp-2 (human larynx epidermoid carcinoma)，利用觀察病毒感染細胞後造成的光學及電子顯微鏡下細胞病變現象、病毒誘發之蛋白質形成、病毒負股RNA之合成及病毒複製能力等實驗方法探討病毒對細胞之喜好性。此外，亦利用微矩陣基因表達分析偵測腸病毒71型感染細胞後，對細胞基因表現的影響。腸病毒71型（100 M.O.I.）感染Vero及SK-N-SH細胞48小時後，會造成嚴重的細胞病變現象，光學顯微鏡下其特徵包括細胞變圓、聚集且最後會死亡。但病毒感染HEp-2及U373MG細胞後，只造成輕微細胞病變現象。腸病毒71型感染Vero及SK-N-SH細胞後，分別誘發14及8條（25-78 kDa）新的蛋白質合成，其中包括病毒的結構蛋白質。而在U373MG及HEp-2細胞中經延長S35-methionine標定時間為6小時後，偵測到1及2條新的蛋白質合成。利用slot blot偵測病毒負股RNA在細胞中的合成情形，發現病毒RNA在所有的細胞中皆可順利複製。以電子顯微鏡觀察細胞內病毒的繁殖情形，發現病毒在所有的細胞株皆有不同程度的繁殖，並以規則的排列方式或不規則的群聚，聚集在細胞質內，顯示病毒能夠順利完成複製及組合過程。此外病毒感染48小時後，Vero及SK-N-SH細胞釋出較多的病毒顆粒（1×108 PFU/ml），而HEp-2及U373MG細胞則釋出較少之病毒(1×105 PFU/ml及1×104 PFU/ml)。利用微矩陣基因表達分析偵測病毒感染SK-N-SH、HEp-2及U373MG細胞的結果顯示，分別有12、7及12個基因受到正反向調控，其中腸病毒71型感染神經細胞後能專一地影響10個基因的表現，包括Oct-1等對於神經生理及存活相關的基因。總之，腸病毒71型能感染所測細胞株，製造RNA、蛋白質最後組合並釋放子病毒，而病毒在神經細胞SK-N-SH中的複製最好。所以我們認為腸病毒71型對神經細胞有特殊的喜好性及致病性，這結論與腸病毒71型感染後所造成的嚴重的臨床病徵可能有密切關係。

[ 英文摘要 ]

Abstract
The clinical syndromes of Enterovirus 71（EV71）infection include hand-foot-mouth disease, herpangina, aseptic meningitis, poliomyelitis-like paralysis and fatal encephalitis. The pathogenesis and tissue tropism of enterovius infection are still poorly understood. Our objectives are to examine EV71 infection in four mammalian cell lines：Vero（Africa green monkey kidney）, SK-N-SH (human neuroblastoma), U373MG (human glioblastoma), and HEp-2 (human larynx epidermoid carcinoma) for clarifying viral tissue tropism. We observed the cytopathic effect (CPE) using phase contrast microscope and electronmicroscope, the synthesis of infected cell specific proteins (ICSPs), viral RNA negative strand synthesis, and virus replication rates in the cells. In addition, we also use microarray assay to examine the cellular genes influenced by EV71 infection. EV71 infection caused obvious CPE at a M.O.I. of 100 in Vero and SK-N-SH cells at 48 h postinfection (p.i.). The cells became rounding-up, aggregated together, and finally died. In contrast, the CPE was very mild in HEp-2 and U373MG cells. The severity of EV71 CPE in various cell lines in general corresponded with ICSPs expression, viral negative strand RNA synthesis and virus replication rates. In the EV71-infected Vero and SK-N-SH cells, 14 ICSPs and 8 ICSPs could be detected, respectively. Their molecular weights were range from 25 kDa to 78 kDa starting at 24 h p.i. One ICSP in U373MG cells and two ICSPs in HEp-2 were detected while the labeling time was extended to 6 h. Slot blot analysis revealed that EV71 could replicate in all cells. The electronmicrographs showed that EV71 could replicate in four cell lines, and caused cell chromatin condensation, vacuolation and cellular surface protusions. Further more, the virus replication rates reached 108 PFU per ml within 48 h p.i. in Vero and SK-N-SH cells and the titer in HEp-2 and U373MG cells were 105 and 104, respectively. Microarray assay data displayed that at least 12、7 and 12 genes were influenced in SK-N-SH、HEp-2 and U373MG cell lines, respectively. Ten genes such as Oct-1 were specially induced in EV71 infected SK-N-SH cell lines. In conclusion, we demonstrated that EV71 infection of SK-N-SH cell showed specifically neurotropism and neurovirulence for the excellence in efficient viral proteins and RNA expression ability in life cycle. This finding may explain the unusual pathogenesis of EV71 infection.