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研究生:孫忠男
研究生(外文):CHUNG NAN SUN
論文名稱:登革二型病毒感染引起內皮細胞及星狀細胞病變之研究
論文名稱(外文):The cytopathogenesis induced by Dengue 2 virus infection in endothelial cells and astrocytes
指導教授:王長君
指導教授(外文):WANG JAANG JIUN
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:108
中文關鍵詞:登革二型病毒內皮細胞星狀細胞細胞病變
外文關鍵詞:Dengue 2 virusendothelial cellastrocytecytopathogenesis
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登革病毒在分類上屬於黃質病毒科,可以引起登革熱、登革出血熱及登革休克症候群。登革出血熱和登革休克症候群除了主要病徵為血管性的外漏現象及出血之外,還包括神經性的病變和腦炎症狀。已有研究報告指出內皮細胞為登革病毒之宿主細胞之一而且在登革休克症候群的嬰兒腦中也發現了病毒但是登革病毒如何進入腦部造成神經細胞功能喪失則屬未知在本實驗中我們利用人類膌靜脈內皮細胞株人類星狀細胞株及初級培養之人類神經細胞來觀察台灣本土登革二型病毒株 PL046 之感染所引起之細胞形態變化及病毒蛋白質定位。登革二型病毒感染的細胞會有顆粒性內質網膨大、粒線體脹大及膜狀小泡構造增生的細胞病變情形出現,並且可以在星狀細胞及神經細胞的膨大顆粒內質網、膜狀小泡與細胞質中找到病毒顆粒。利用免疫螢光染色及免疫膠基金的方式標定病毒蛋白質 E 及 NS1,發現此二種病毒蛋白質均表現在細胞核周圍。利用 TUNEL 染色亦得知登革病毒感染會引起內皮細胞之細胞凋亡現象。根據這些結果,我們推測登革病毒感染所引起的神經細胞病變可能是經由內皮細胞及星狀細胞的感染所致。
Dengue virus is a member of the flaviviridae and causes dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The vascular leakage and hemorrhagic diathesis are the hallmarks of the DHF/DSS, including neurological disorders and encephalitis. It has been reported that endothelial cells may be one of the targets of dengue virus. Dengue virus was also detected in the human brain of a fatal case of DHF, but whether dengue viruses penetrate to brain and cause neuron defects is still unknown. In this study, human umbilical vein endothelial cell line ECV304, human astrocyte cell line HTB-17 and human neuron primary culture were infected with a local isolate strain of DEN-2, PL046, to detect the morphological changes and the viral protein localization. Cytopathological changes, swelled rER and enlarged mitochondria, were observed in cells infected with DEN-2. Virus particles were found in the swelled rER of human neuron primary culture and HTB17.In immunofluorescein and immunogold labeling experiments, ECV304 and HTB-17 show positive results. Viral proteins NS1 and E were localized in the perinuclear regions. DEN-2 infection induced apoptosis was also observed in ECV304 cells by TUNEL methods. According to these results, DEN-2 infection may cause neuron defects via endothelial cells and astrocytes.
頁次
中文摘要………………………………………………………… V
英文摘要…………………………………………………………VI
緒言 ………………………………………………………………1
材料與方法…………………………………………………………9
壹、 材料 …………………………………………………………9
一、 實驗所用細胞株 …………………………………………9
二、 實驗所用抗體 ……………………………………………10
三、 實驗使用之病毒株…………………………………………11
四、 各項溶液之配法……………………………………… 11
五、 實驗儀器………………………………………………13
六、 實驗所用藥品之公司與出產國 ……………………………14
貳、 實驗方法……………………………………………………17
一、細胞培養 ……………………………………………………17
二、病毒感染 ……………………………………………………17
三、病毒力價的測定 ……………………………………………18
四、細胞染色 ……………………………………………………19
(一)、免疫螢光染色 ……………………………………19
(二)、細胞核之染色 ……………………………………20
(三)、利用 TUNEL 試劑來偵測進行細胞凋亡之細胞…20
五、電子顯微鏡樣品之處理………………………………………21
(一)、電子顯微鏡觀察細胞形態之標本製備 …………21
(二)、包埋後進行免疫細胞化學之標本製備 …………21
(三)、平坦包埋 …………………………………………22
(四)、超薄切片 ………………………………………22
(五)、包埋後免疫膠基金之標定 ……………………23
(六) 雙重染色 ………………………………………23
結果…………………………………………………………………25
壹、 光學顯微鏡之觀察…………………………………………25
一、 ECV304細胞之免疫螢光化學染色…………………………26
二、 HTB-17 細胞之免疫螢光化學染色 ………………………26
三、 細胞凋亡的觀察 ……………………………………………27
貳、 電子顯微鏡之觀察…………………………………………28
一、 ECV304 細胞感染登革二型病毒後之超微結構變化………28
二、 病毒蛋白質NS1及E在受感染的ECV304細胞中的表現位置
標定 ………………………………………………29
三、 HTB-17 細胞感染登革二型病毒後之超微結構變化………30
參、 病毒力價測定………………………………………………31
一、 登革二型病毒在ECV304細胞上清液中之力價測定 ……… 31
討論…………………………………………………………………32
參考文獻……………………………………………………………32
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