感染廣東血線蟲鼷鼠腦部免疫反應指標表現之時間序列變化研究

Time course change in expression of immune response markers of mouse brain infected with Angiostrongylus cantonensis

王永裕(Yung-Yu Wang)

指導教授 ： 蘇霩靄

國立臺灣大學/醫學院/微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02147

摘要

廣東血線蟲 (Angiostrongylus cantonensis) 為台灣重要的人畜共通性寄生蟲。當人食入廣東血線蟲感染性幼蟲，幼蟲鑽入腸壁隨循環系統進入腦部，幼蟲在此發育為幼成蟲並可能導致嗜酸性腦膜腦炎。他種哺乳動物如鼷鼠、兔等可作為人類廣東血線蟲的動物模式。前人研究顯示實驗感染廣東血線蟲的鼷鼠，其CSF中含大量的Th2-type細胞激素 (如IL-4與IL-5)，我們以RT-PCR亦證明在感染14天時腦實質中會表現大量的Th2-type細胞素。但對感染後腦內免疫反應連續變化之資訊較為欠缺。本研究利用RT-PCR、H&E染色與免疫化學染色來探討鼷鼠在感染廣東血線蟲後，其腦組織中各種細胞素之變化，藉此直接探討腦部免疫反應之演繹。實驗中餵食30隻L3幼蟲予ICR鼷鼠，並於感染後10、11、12、13、14天犧牲鼷鼠取腦。切片顯示鼷鼠腦部出現點狀出血的時間大概在感染後第9~10天，而出血可能與蟲體的移行有關。但經HE染色得到發炎細胞大量進入腦部第三腦室的時間為第13天。以RT-PCR觀察助發炎細胞激素 (IL-1β與TNF-α)、Th1/Th2細胞激素 (IFN-γ及IL-5) 與神經滋養因子 (NGF、BDNF與NT-3)基因之表現，發現IL-1β、TNF-α與Th1細胞素IFN-γ在第10天開始即有明顯表現。TNF-α與IL-1β在第12天之後的表現量大增而於第13天達到高峰。Th1細胞素IFN-γ在第11天稍增，12-13 天之表現最強而後減弱；IL-5之基因表現第11-14天明顯增加。NGF與NT-3並沒有明顯的改變，但是BDNF從第10到12天之間卻有明顯的增加，第13天降至接近未感染組，但第14天又再提升。助發炎細胞激素可影響腦內的通透性，使大量的發炎細胞得以進入。IFN-γ與發炎反應的擴大亦有關連。從腦部開始發生出血斑點到大量發炎細胞的進入之間的時間差異，腦部或許有延緩發炎反應的作用，而神經滋養因子與Th2細胞激素都可幫助神經細胞生存。神經滋養因子在嗜伊紅性腦膜腦炎的初期，或許有延緩發炎反應擴大的效果。雖然發炎反應持續擴大，神經細胞藉分泌神經滋養因子與Th2細胞激素在發炎反應下延續其生命。

關鍵字

廣東血線蟲 ； 助發炎細胞激素 ； 丙型干擾素 ； 間白素-五 ； 神經滋養因子

並列摘要

Angiostrongylus cantonensis is an important zoonotic parasite in Taiwan.
When infective larvae are ingested by human, they enter the blood vessels of the intestinal tract and eventually reach the brain to develop into young adult worms and cause eosinophilic meningoencephalitis. Other mammals like mouse and rabbits serve as animal models for human angiostrongyliasis. Previous studies revealed that cerebrospinal fluid contained high level Th2 cytokines like IL-4 and IL-5 in infected mice. We also observed the expression of Th2-type cytokines in mouse parenchyma at 14 days postinfection (dpi). In this study, we used RT-PCR, H&E stain and immunohistochemistry techniques to monitor the time-course change in cytokines in the brain, to understand the change in immune response in the brain of infected mouse. ICR mice were each orally infected with 30 infective larvae and sacrificed at 10,11,12,13 and 14 dpi. Section of the brains revealed that spotted hemorrhage appeared in the mouse brain at 9-10 dpi, possibly related to migration of the worms. Leukocytes infiltration into the third ventricle was more drastic thanthat of the meninge at 13 dpi. The gene expression of TNF-α, IL-1β and the Th1 cytokine-IFN-γ started to elevate at 10 dpi. TNF-α and IL-1β expression reached their peak on 13 dpi. IFN-γ expression increased slightly on 11 dpi and peaked 12-13 dpi. IL-5 expression increased significantly during 11 to 14 dpi. No apparent change in expression of NGF and NT-3 were observed. BDNF expression, however, increased significantly between 10-14 dpi except at 13 dpi which was almost normal. Proinflammatory cytokines may affect the permeability in the brain and resulted in leukocyte infiltration. The brain might be able to postpone inflammatory response after hemorrhage occurred in the brain, hence cellular infiltration occurred a few days afterward. Neurotrophic factors and Th2 cytokines are known to help neuron survival. Neurotrophic factor might have the effect of retarding inflammatory response in early eosinophilic meningoencephalitis cause by Angiostrongylus cantonensis infection in mice. Thus, although inflammation response kept on expanding, neuronal cells could secrete neurotrophic factors and Th2 cytokines to sustain their life in the presence of inflammatory response.

並列關鍵字

Angiostrongylus cantonensis ； proinflammatory cytokines ； IFN-gamma ； IL-5 ； neurotrophic factors

參考文獻

Alicata, J.E., Jindrak, K. 1970. Angiostrongylosis in the Pacific and Southeast Asia. Charles C.Thomas Pub., Springfield, Ⅲ.

Allan, S.M., Rothwell, N.J. 2001. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2, 734-744.

Alvarez, J.I., Teale, J.M. 2007. Differential changes in junctional complex proteins suggest the ependymal lining as the main source of leukocyte infiltration into ventricles in murine neurocysticercosis. J Neuroimmunol 187, 102-113.

Aoki, M., Sugaya, H., Ishida, K., Yoshimura, K. 1998. The role of CD4(+) and CD8(+) T-cells in host morbidity and innate resistance to Angiostrongylus cantonensis in the mouse. Parasitol Res 84, 91-99.

Argaw, A.T., Zhang, Y., Snyder, B.J., Zhao, M.L., Kopp, N., Lee, S.C., Raine, C.S., Brosnan, C.F., John, G.R. 2006. IL-1beta regulates blood-brain barrier permeability via reactivation of the hypoxia-angiogenesis program. J Immunol 177, 5574-5584.