第二型豬環狀病毒誘發肉芽腫病變中細胞激素表現與病毒之相關性研究

中文摘要本實驗主要目的在探討PCV2 感染豬隻所引發之組織球浸潤及多核巨細胞形成等肉芽腫性病變之病理機制。首先以豬肺臟巨噬細胞及豬周邊血液單核細胞為研究材料，與PCV2 病毒於試管內模式下進行長時間的培養，觀察其型態學的改變，偵測上清液細胞激素如：interleukin (IL)-1β，IL-8 及IL-10 的表現量，並且監測感染後細胞之存活率。同時採取現場PCV2 感染之PMWS 豬隻的淋巴結，經由病理切片判讀後將病變分類以進行免疫化學染色，來偵測淋巴結中，interferon (IFN)-γ，IL-1β，IL-8 及IL-10 等細胞激素的表現情形；並以原位雜交法偵測病毒核酸反應，以比較病變、細胞激素表現量及分佈，與病毒核酸分佈三者之間的相互關係。結果在試管內模式下，豬肺臟巨噬細胞及豬周邊血液單核細胞的上清液中可發現細胞激素IL-8 的大量表現，在豬肺臟巨噬細胞的上清液也發現IL-1β的表現量較實驗組略為增加。在長時間的培養下，同時發現PCV2 感染後，細胞存活率有下降的情形。在細胞激素免疫化學染色中，發現PMWS豬隻淋巴結內有大量IFN-γ與IL-1β之陽性訊號。比較四種病變包括組織球浸潤、嗜中性球浸潤、組織球與嗜中性球同時存在，和病變輕微淋巴結的細胞激素以及病毒核酸的分佈後，發現IFN-γ的陽性細胞與組織球浸潤的病變之分佈區域相近，皆在淋巴濾泡生發中心區與副皮質區域，組織球病變中PCV2 之病毒量也較高。IL-1β則與嗜中性球浸潤有位置相關性。但細胞激素的表現分佈似乎與病毒核酸的分佈並無空間相關性。在各種病變的細胞激素表現量之比較，發現組織球浸潤病變的IFN-γ的陽性細胞表現量較其他各組為高；嗜中性球浸潤病變之IL-1β的陽性表現量與其他各實驗組相較也呈顯著的增加。因此，PCV2 的感染可能造成IFN-γ分泌，進而誘發肉芽腫性反應的產生。

關鍵字

肉芽腫性病變；豬環狀病毒；細胞激素

並列摘要

The main purpose of this study was to investigate the pathogenesis between PCV2 infection and PMWS lesions, namely histiocytosis or granulomatous lesions. Alveolar macrophages (AM) and peripheral blood monocytes (PBM) were obtained from 5 to 10-week-old healthy pigs free of PCV2 and PRRSV infection. The AM and PBM were inoculated with PCV2 and the viability, infectious rate, and morphological changes were observed and recorded. The supernatants were collected and the cytokine profiles were examined using commercial ELISA kits. Meanwhile, paraffin-embedded lymphoid tissues of field PMWS cases, which were negative in PCR results of pseudorabies virus, PRRS virus, and classic swine fever virus, were collected and graded for different stages of granulomatous lymphadenitis. Serial sections were made and detected later by in situ hybridization for the distribution of PCV2 viral nucleic acid. Immunohistochemical stainings for several granulomatous lesion-related cytokines, such as IL-1β, IL-8, IL-10, and IFN-γ, were performed. The results revealed that AM and PBM produced high levels of IL-1β and IL-8 in response to PCV2 infection, compared with non-infected cells. The significant effect of PCV2 infection on these cells started at 24 hours post infection.. In the 192 hours of incubation, the decrease in viability of PAM and PBM were also noted. The results of immunohistochemistry staining for cytokines revealed high expression of IFN-γ and IL-1β in the lymph nodes of PMWS pigs, compared with the control SPF pigs. The expression of IFN-γ was strong in the lymph nodes accompanied with lesions of histiocytosis. The expression of IL 1-β was strong in the lymph nodes accompanied with lesions of granulocytic infiltration. The distribution of PCV2 seemed to have no correlation with the distribution of cytokine signals. However, the viral loadings were strong in lymph nodes with histiocytosis lesions. The immnunohistochemistry staining result of IL-8 revealed minimal to negative pattern compared with the results of in vitro system, while IL-10 results were both negative in the in vitro system and IHC. In conclusion, PCV2 infection might enhance IFN-γproduction and thus trigger the granulomatous reactions.

並列關鍵字

grnulomatous ； PMWS ； PCV2 ； cytokine

參考文獻

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第二型豬環狀病毒誘發肉芽腫病變中細胞激素表現與病毒之相關性研究

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