| 摘要: | 廣東住血線蟲（Angiostrongylus cantonensis）會感染終宿主大鼠的心臟和肺動脈，引起體內病變。在台灣廣東住血線蟲常以非洲大蝸牛和福壽螺作為其中間宿主，人類和小鼠同樣都是廣東住血線蟲的非適當宿主，若誤食經廣東住血線蟲感染的螺或生食，就有可能因為食入幼蟲而導致蟲體移行至腦及脊隨處，進而造成嚴重的嗜伊紅性腦膜炎（eosinophilic meningitis）或腦膜腦炎（meningoencephalitis）。
本研究使用18F-FDG PET/CT正子成像模型使本研究能夠在宿主整個發病過程中直接觀察活體內的腦部病變．實驗中使用18F-FDG作造影示蹤劑，檢測BALB/c 及C57BL/6小鼠經廣東住血線蟲感染後腦部各個區域的發炎情形，並配合利用免疫組織學（IHC）及qRT-PCR分子生物技術實驗，分別找出在轉錄與轉譯層次在不同腦區扮演重要角色的免疫及發炎因子。
實驗結果顯示：BALB/c小鼠經由PET照影在18F-FDG的吸收趨勢在大腦各區域均隨著感染時間而增加，反觀C57BL/6小鼠則在感染第二週到第三週略為增加，到第四週吸收量下降．而在qRT-PCR及IHC中，作為Th2嗜伊紅性白血球趨化因子的Ym1在BALB/c小鼠中，腦部各區域大致上都隨著感染時程的延長有更高的表現；另外作為小膠質細胞和巨噬細胞的鈣離子結合性蛋白的Iba1，在qRT-PCR中無論在受到感染的BALB/c小鼠還是C57BL/6小鼠腦部各區域都會有不同程度的聚集表現，在感染二週到三週期間大量聚集，感染到第四週時則下降；在IHC中C57BL/6小鼠的趨勢和在qRT-PCR中相似，而BALB/c小鼠的表現量則偏低．此外透過qRT-PCR也得知Th2相關之細胞激素，例如：IL-4、IL-5、IL-6、IL-13、IL-33等，以及免疫反應相關的細胞激素，例如：IFN-γ和c-fos，會參與廣東住血線蟲引發嗜伊紅性腦膜炎相關的腦部發炎反應．而透過上述的實驗數值媒合也發現，BALB/c小鼠經廣東住血線蟲感染第三週到第四週在Ym1的表現量和18F-FDG的吸收趨勢有較高的相關性，而BALB/c小鼠經廣東住血線蟲感染第二週到第三週在Iba1的表現量則和18F-FDG的吸收趨勢在有較高的相關性。
綜合以上結果顯示，Ym1對於廣東住血線蟲感染後期具有顯著的影響力，經由PET/CT及qRT-PCR實驗數值的相關係數媒合計算，可以應用18F-FDG-PET活體動物影像的評估模式來檢測廣東住血線蟲感染引起宿主腦部發炎的病理損傷變化。而其中小膠質細胞和巨噬細胞也參與由廣東住血線蟲感染引起的早期腦部發炎，影響非適當宿主小鼠腦部各區域中的免疫病理機制。本研究結果可望在未來應用於嗜伊紅性腦膜炎或腦膜腦炎的臨床影像的病理診斷上。
Angiostrongylus cantonensis often infects the heart and pulmonary arteries of the definitive host, rat, causing internal diseases. Angiostrongylus cantonensis living in Taiwan often use Achatina fulica and Ampullaria canaliculate as their intermediate hosts. Humans and mice are also non-permissive hosts for Angiostrongylus cantonensis. If humans accidentally eat snail or raw food infected with Angiostrongylus cantonensis, it may cause the worms to migrate to the meninges due to the accidental eating of larvae, which may cause severe eosinophilic meningitis or meningoencephalitis.
18F-FDG PET/CT imaging model used in this study allows us to directly observe the host’s brain lesions in vivo during the disease process, using 18F-FDG as PET tracer to detect BALB/c and C57BL/6 mice inflammation in various brain regions after Angiostrongylus cantonensis infection during the research, and using immunohistochemistry (IHC) and molecular biology (qRT-PCR) related experiments to find out the inflammatory factors that play an important role in the level of transcription and translation in different brain regions.
Results showed that the trends of 18F-FDG uptakes in BALB/c mice through PET imaging increased with the infection time in all regions of the brain, while C57BL/6 mice increased slightly from the second to the third week post-infection, uptakes decreased by the fourth week; and in qRT-PCR and IHC, Ym1, a Th2 eosinophilic chemotactic factor, which were gradually enhanced and reached to peak in all regions of brain in BALB/c mice. Iba1, ionized calcium-binding protein of microglia and macrophage, no matter if it is infected in BALB/c mice or C57BL/6 mice, there will be different level accumulation in various areas of the brain, which were gradually enhanced from the second to the third week post-infection, decreased by the fourth week in qRT-PCR. In IHC, the trends of C57BL/6 mice were similar to qRT-PCR, while BALB/c mice were in low expression. In addition, Th2 related cytokines in qRT-PCR, such as IL-4, IL-5, IL-6, IL-13, IL-33 etc., and other immune response related cytokines, such as IFN-γ and c-fos, will participate in the brain inflammation related to eosinophilic meningitis which triggered by Angiostrongylus cantonensis, and through the matching of the above experimental data, it is also found that there was a high correlation between the expression level of Ym1 and the trends of 18F-FDG uptakes in BALB/c mice infected by Angiostrongylus cantonensis from the third week to the forth week, and a high correlation between the expression level of Iba1 and the trends of 18F-FDG uptakes in BALB/c mice infected by Angiostrongylus cantonensis from the second week to the third week.
Collectively, these results suggest that Ym1 plays an important role in the late stage of infection. Through the calculation of the correlation coefficient of the PET/CT and qRT-PCR, the 18F-FDG-PET animal imaging model with Ym1 can be used to detect the pathological damage of the host’s brain inflammation caused by the infection of Angiostrongylus cantonensis. Microglia and macrophage may also participate the early brain inflammation caused by Angiostrongylus cantonensis, to influence the immunopathogenesis in various regions of the brain of non-permissive host, mice. The results in the present study is expected to be integrated in the future, applying in the clinical treatment of eosinophilic meningitis or meningoencephalitis. |
