- 學位論文
類鐸接受體與細胞激素於豬生殖與呼吸綜合症所扮演的角色
Roles of toll-like receptors and cytokines in the development of porcine reproductive and respiratory syndrome
摘要
在養豬產業中，豬生殖與呼吸綜合症（porcine reproductive and respiratory syndrome; PRRS）是嚴重影響其經濟效益的病毒性疾病之一。此病臨床呈現流產和呼吸症狀，造成哺乳豬和離乳豬死亡與發育遲緩。本實驗以PRRS病毒（ PRRS virus; PRRSV ）感染豬隻後應用半定量RT-PCR檢測體內類鐸接受體（Toll-like receptors; TLRs）mRNA之表現量，並利用商業化酵素免疫粘著吸附 (ELISA）套組檢測細胞激素之濃度，探討PRRSV的可能免疫及致病機制。受PRRSV感染之豬隻其肺門淋巴結TLR3之mRNA表現量於感染後第7天低於未感染豬隻，但未達顯著水準 ( P = 0.11 )，而 TLR7則於感染後第14天，其mRNA表現量顯著 ( P < 0.05 ) 高於未感染豬隻；此外，PRRSV 感染豬隻其脾臟TLR8之mRNA表現量於感染後第14天高於未感染豬隻，但未達顯著水準 ( P = 0.16)。周邊血液單核細胞（peripheral blood mononuclear cells; PBMCs）之TLR2、TLR4及TLR8 mRNA表現量於PRRSV感染後第7天顯著 ( P < 0.05 ) 高於未感染豬隻，TLR3及TLR7亦上升，但未達顯著水準 ( P = 0.12及0.08 )。豬隻受PRRSV 感染後14天，其肺泡沖洗液之IL-1、IL-8、IL-12、TNF-α及IFN-γ之表現量較未感染豬隻高，但皆未達顯著水準 ( P = 0.13-0.23 )。與未感染豬隻相較，PRRSV 感染豬隻其肺泡巨噬細胞（alveolar macrophages; AMs）及PBMCs以PRRSV及病原體相關分子模式刺激後，其IL-1、IL-6、IL-12及TNF-α表現量於許多組別出現顯著 ( P < 0.05 ) 上升。上述結果顯示，豬隻受PRRSV 感染後其體內TLRs表達 上升，此可能與豬隻罹患PRRS後易受二次性感染而加重病情有關。而肺泡沖洗液中IL-1及TNF-α表現量上升可能與豬隻罹患PRRS後之臨床症狀有關。曾感染PRRSV豬隻，其AMs及PBMCs以PRRSV刺激後之IL-12上升顯示細胞免疫反應已經受到活化，而IL-1、IL-6及TNF-α的表現上升，可能與臨床症狀的惡化有關。
並列摘要
Porcine reproductive and respiratory syndrome（PRRS）, one of the most economically significant viral diseases in the swine industry, is characterized by abortions and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. To elucidate the possible immunological and pathological mechanisms induced by PRRS virus ( PRRSV ) in pigs, the expressions of toll-like receptors ( TLRs ) and cytokines in PRRSV challenged pigs were determined. The TLRs mRNA expression was measured by semi-quantitative RT-PCR and cytokine concentrations were determined using commercial enzyme-linked immunosorbent assay ( ELISA ) kits. The IL-1, IL-8, IL-12, TNF-α and IFN-γ expressions in bronchoalveolar lavages were higher than those of uninfected pigs at 14 days post-infection ( dpi ), but not at a significant level ( P = 0.13-0.23 ). The increased IL-1 and TNF-α expressions in bronchoalveolar lavages might be relevant to clinical symptoms in PRRSV infected pigs. The TLR3 mRNA expression in tracheobronchial lymph nodes in PRRSV infected pigs was lower than that of uninfected pigs at 7 dpi, but not at a significant level ( P = 0.11 ) . The TLR7 mRNA expression in tracheobronchial lymph nodes in PRRSV infected pigs was significantly ( P < 0.05 ) higher than that of uninfected pigs at 14 dpi. The TLR8 mRNA expression in spleen in PRRSV infected pigs was higher than that of uninfected pigs at 14 dpi, but not at a significant level ( P = 0.16 ). The TLR2, TLR4 and TLR8 mRNA expressions in peripheral blood mononuclear cells ( PBMCs ) in PRRSV infected pigs were significantly ( P < 0.05 ) higher than those of uninfected pigs at 7 dpi. The TLR3 and TLR7 mRNA expressions were higher than those of uninfected pigs, but not at a significant level ( P = 0.12 and 0.08, respectively ). The IL-1, IL-6, IL-12 and TNF-α expressions in PRRSV and pathogen associated molecular patterns ( PAMPs ) stimulated alveolar macrophages ( AMs ) and PBMCs from PRRSV infected pigs were most often observed to be significantly ( P < 0.05 ) increased as compared with those of uninfected pigs. Our results indicate that the increased expressions of TLRs in PRRSV infected pigs might be associated with higher susceptibility to secondary infections and thus with more severe clinical outcomes. The up-regulated IL-12 in PRRSV- and PAMP- stimulated AMs and PBMCs from PRRSV infected pigs suggested that the immune system was activated after viral infection. The increased IL-1, IL-6 and TNF-α might be correlated with the severe clinical signs induced by PRRSV and secondary invaders.