探討登革病毒在Stat1剔除鼠皮膚血管內皮細胞功能之影響

The kinetic change in the expression of functional proteins in skin endothelial cells of dengue hemorrhage in Stat1-/- mice

鄭敏貞(Min-Jhen Jheng)

指導教授 ： 伍安怡

國立臺灣大學/醫學院/免疫學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703915

摘要

登革病毒是世界上重要的蟲媒傳染性疾病之一。具有登革出血熱(DHF)或登革休克症候群(DSS)的患者相對於登革熱(DF)患者有較高的死亡率。藉由登革出血小鼠模式動物，浸潤到皮下組織的巨噬細胞會產生TNF且內皮細胞會有細胞凋亡的現象。在這個研究中，將以出血小鼠的皮膚及登革感染後的內皮細胞探討關於內皮細胞的黏著分子(adhesion molecules)、趨化因子(chemokines)及緊密連接蛋白(tight junction proteins)等特徵在登革病毒刺激後所產生的變化。在登革病毒感染後第五天會在Stat1剔除鼠的皮膚觀察到出血現象。透過抗體陣列分析，M-CSF、CCL2、CCL3、CCL5及CXCL2的表現量在病毒感染後都會顯著提升。藉由免疫螢光染色的方式，在病毒感染後第三天在出血皮膚上便可以看到大量的巨噬細胞浸潤的情況，且可以觀察到內皮細胞在病毒感染後第五天會表現高量的ICAM-1。從皮膚亦可以觀察到緊密連接蛋白ZO-1及occludin皆會受到病毒影響而有所改變。在體外實驗系統的研究中，登革病毒會造成內皮細胞HUVEC或HMEC-1及硫乙醇酸(thioglycollate)在Stat1剔除鼠所引發的巨噬細胞產生巨噬細胞的趨化因子，且受登革感染的巨噬細胞上亦會表現趨化因子受體CCR2。雖然以重組蛋白TNF或登革病毒直接刺激HMEC-1只能造成ICAM-1的低度表現，但兩者的組合下便能顯著提升ICAM-1的表現量。再者，登革病毒會促使HMEC-1 的occludin表現量下降且ZO-1被切割後的片段會釋出到胞外，由此可推斷登革病毒可能是透過破壞內皮細胞間的緊密連接蛋白而改變其通透性。接著我們探討了C型肝炎病毒蛋白酶抑制劑(asunaprevir)是否會對登革感染及出血產生影響。Stat1剔除鼠在感染登革病毒後以管餵的方式連續投藥，asunaprevir會減少脾臟中的病毒含量及促發炎細胞因子Tnf的表現量，且在腹部皮膚上的出血情況也有減緩的趨勢。
這個研究顯示登革病毒感染會造持內皮細胞產生功能性改變，包含黏著分子、趨化因子的增加及緊密連接蛋白的改變。這些在內皮細胞上產生的變化對於出血發生可能扮演著舉足輕重的腳色。另外在小鼠身上進行asunaprevir的藥效探討則提供了針對登革病毒感染上老藥新用的發展契機。

關鍵字

登革病毒 ； 登革出血 ； 黏著分子 ； 緊密連接蛋白 ； 巨噬細胞浸潤

並列摘要

Dengue virus (DENV) is one of the most important vector-borne infectious diseases in the world. Patients with dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS) have much higher mortality than those with dengue fever (DF). We established a hemorrhage mouse model in Stat1-/- mice by inoculating 106 PFU of DENV intradermally. Stat1-/- mice developed hemorrhage on day 5 after infection. By using antibody array, we discovered that M-CSF, CCL2, CCL3, CCL5 and CXCL2 were up-regulated in the skin on day 5 after infection when hemorrhage developed. Immunofluorescence staining of skin cryosections revealed that ICAM-1 was upregulated on CD31+ endothelial cells and macrophage infiltration was detected as early as day 3 after infection. Western blotting analysis of homogenized skin tissues showed that tight junction proteins ZO-1 and occludin were altered when hemorrhage developed.
In vitro studies showed that DENV infection induces HUVEC, HMEC-1 and thioglycollate-elicited Stat1-/- macrophages to produce macrophage chemoattractants and that macrophages expressed chemokine receptor CCR2. While rhTNF and DENV separately induced marginal expressions of ICAM-1 in HMEC-1, DENV combined with TNF treatment significantly enhanced ICAM-1 expression. Moreover, DENV infection increased the release of cleaved ZO-1 in HMEC-1. These results suggest that DENV infection causes tight junction disruption thereby changing the permeability of endothelial cells.
To test the effect of asunaprevir, DENV-infected Stat1-/- mice were given asunaprevir intragastrically at the time of and after infection. Results showed that asunaprevir reduced viral load and Tnf transcripts in the spleen as well as the severity of hemorrhage development in the abdominal skin compared to vehicle controls. These data showed that repurposing asunaprevir for treatment of dengue is potentially a feasible approach.
This study revealed that DENV infection resulted in functional changes of endothelial cells which may be crucial to hemorrhage development. Moreover, we showed that Stat1-/- mice are useful in testing the efficacy of potential anti-DENV drugs.

並列關鍵字

Dengue virus ； Dengue hemorrhage ； adhesion molecules ； tight junction proteins ； macrophage infiltration ； asunaprevir

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