小鼠之內皮細胞對於登革熱病毒感染而引發自 然凋亡的機制探討

Dengue virus induces murine microvascular endothelial cell apoptosis

黃郁華(Yu-Hwa Huang)

指導教授 ： 伍安怡

國立臺灣大學/醫學院/免疫學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10202

摘要

有鑒於登革熱病毒感染會造成內皮細胞的死亡，一直被認為是造成登革出血熱的相關因子;雖之如此，從來沒有實驗研究是否登革熱病毒會引發自然凋亡(Apoptosis)。在此論文中，我提出這樣的假設，以老鼠與人類內皮細胞為材料來探討此課題。
我從小鼠的腦組織純化出的微小血管內皮細胞，其表面是帶有CD31 分子標記以及表現內皮細胞一氧化氮生成酵素(eNOS)的。我發現小鼠內皮細胞是登革第二型出血性病毒寄主細胞，因觀察到病毒可以感染與複製於內;並且可以引發其表現誘發型一氧化氮生成酵素(iNOS)，然後引起細胞自然凋亡的現象。然而，在加入誘發型一氧化氮生成酵素抑制化學物質(L-NAME)會降低其自然凋亡的現象，同樣的，在一氧化氮生成酵素基因缺陷小鼠內皮細胞亦可觀察到;另外，登革熱病毒的感染會引起小鼠內皮細胞產生高量的一氧化氮，所以推測一氧化氮可以是造成登革熱病毒的感染而導致內皮細胞自然凋亡的重要分子。
此外，以登革熱病毒感染人類大血管內皮細胞(HUVEC)以及人類轉型微小血管內皮細胞株(HMEC)結果顯示這些細胞會被引起自然凋亡。若加入一氧化氮抑制劑(L-NAME)以及活化氧激分子抑制劑(NAC)則可部分抑致自然凋亡的結果。令人驚訝的是，若再同時加入此兩種抑制劑時，自然凋亡的現象可以完全抑致其凋亡現象。結果顯示，一氧化氮以及活化氧基分子兩者都參與引發細胞凋亡。此外，我更發現在加入caspase 抑制劑時可以抑致登革熱病毒所引起的自然凋亡現象，這
證實了登革熱病毒所引起的自然凋亡現象是caspase 依賴性的。
總結上述實驗證明登革熱病毒會感染小鼠以及人類內皮細胞，而且引起高量之一氧化氮產生，在透過與活化氧激分子的合成作用之下，造成內皮細胞自然凋亡的現象。結果顯示，自然凋亡發生在登革病毒感染的內皮細胞的現象，與登革出血熱的致病機制，具有關聯性; 此外，由實驗數據顯示，以老鼠內皮細胞感染登革熱病毒所引發的反應是可以與人類細胞類似的，由此可見，用小鼠模式研究登革熱的致病機制的可行性。

關鍵字

登革熱病毒, 內皮細胞, 自然凋亡

並列摘要

Endothelial cell death is suggested as a mechanism by which dengue virus causes hemorrhage, the hallmark of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). It has never been examined whether dengue virus causes endothelial cell death. The present study was designed to address this question in mouse as well as human endothelial
cells. Mouse primary microvascular endothelial cells were isolated from collagenase-treated mouse brain tissue. The isolated cells expressed CD31 and endothelial nitric oxide synthase (eNOS). The results showed that dengue virus serotype 2 strain 16681 established a productive infection in primary mouse microvascular endothelial cells, induced
inducible nitric oxide synthase (iNOS) and apoptotic cell death. Use of NOS inhibitor, n-nitroso-L-arginine methyl ester (L-NAME) reduced dengue virus-induced apoptosis. Dengue virus-induced apoptosis was also reduced in endothelial cells isolated from iNOS-/- mice. These
observations indicated that high output NO was involved in denguevirus-induced endothelial cell apoptosis.
HUVEC, primary human macrovascular endothelial cells, and
HMEC, transformed human microvascular cell line infected with dengue virus like that in primary mouse endothelial cells also underwent apoptosis. Virus-induced apoptotic cell death was partially reduced by treatment with either L-NAME or reactive oxygen species (ROS) scavenger N-acetyl cysteine alone and completed inhibited by treatment
with both inhibitors. These results indicated that both NO and ROS were involved in causing dengue virus-induced endothelial cell damage.
Moreover, zVAD-fmk (pan-caspase inhibitor) prevented dengue-virus induced-apoptosis, demonstrating that dengue virus-induced endothelial cell death was caspase-dependent.
Taken together, results in this study showed that dengue virus infects mouse as well as human endothelial cells. The infection induces high output of NO. Through the combined effect of NO and ROS, dengue virus induces endothelial cell death. These results point out a potentia relationship between dengue virus-induced endothelial cell death and
dengue pathogenesis. In addition, interaction between dengue virus and mouse endothelial cells modeled that between the virus and human cells, suggesting a possibility of using mouse model to study dengue pathogenesis.

並列關鍵字

Dengue Virus, Endothelial cell, apoptosis

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