本文關鍵詞： 抗菌肽Sublancin 產氣莢膜梭菌 耐甲氧西林金黃色葡萄球菌 天然免疫 小鼠　出處：《中國農業(yè)大學》2017年博士論文　論文類型：學位論文

【摘要】：本論文通過四個試驗,探討了抗菌肽Sublancin對肉雞壞死性腸炎的治療效果及其對小鼠天然免疫的影響。(1)試驗一在體外試驗發(fā)現(xiàn)抗菌肽Sublancin對產氣莢膜梭菌CVCC2027具有一定抑菌作用的基礎上,選取252只1日齡愛拔益加肉公雞,隨機分為六個處理,分別為正常對照、產氣莢膜梭菌攻毒對照、3個Sublancin飲水治療組(2.88、5.76和11.52 mg/L)和林可霉素飲水治療組(75 mg/L),試驗期為28 d。在d]5-21對肉雞進行產氣莢膜梭菌經口攻毒,之后進行持續(xù)7 d的Sublancin或林可霉素飲水治療。結果表明,Sublancin緩解了產氣莢膜梭菌攻毒造成的腸道絨毛損傷,降低了回腸促炎性細胞因子IL-1β、IL-6和TNF-α的表達量(P0.05),可達到與林可霉素相似的治療效果。(2)試驗二以巨噬細胞系RAW264.7為體外模型,研究Sublancin對巨噬細胞的活化作用及分子機制,并用小鼠腹腔巨噬細胞作為原代細胞驗證巨噬細胞系得到的試驗結果。結果表明,Sublancin顯著促進了 RAW264.7和小鼠腹腔巨噬細胞IL-1β、IL-6、TNF-α和NO的產生(P0.05),且上調了趨化因子(IL-8和MCP-1)和共刺激分子(B7-1和B7-2)的基因表達(P0.05)。Sublancin顯著增強了巨噬細胞的吞噬功能和對耐甲氧西林金黃色葡萄球菌的殺菌能力,MAPK和NF-κB信號通路參與了 Sublancin對巨噬細胞的活化過程。(3)試驗三分別以正常小鼠、環(huán)磷酰胺免疫抑制小鼠和耐甲氧西林金黃色葡萄球菌感染小鼠為模型,研究Sublancin對小鼠天然免疫的影響,揭示Sublancin增強小鼠抗金黃色葡萄球菌感染能力的機理。試驗結果表明,正常小鼠中,灌服Sublancin提高了小鼠腹腔巨噬細胞的吞噬功能(P0.05),并顯著上調了小鼠腹腔巨噬細胞IL-1β、IL-6和TNF-α的基因表達(P0.05)。在環(huán)磷酰胺免疫抑制小鼠中,Sublancin緩解了環(huán)磷酰胺對小鼠巨噬細胞吞噬活性的抑制作用,緩解了環(huán)磷酰胺造成外周血中紅細胞、血紅蛋白、白細胞和血小板含量的降低。與環(huán)磷酰胺模型組相比,抗菌肽Sublancin(4.0和8.0 mg/kg體重)治療后,顯著上調了小鼠脾臟中IL-2、1L-4和IL-6的基因表達(P0.05)。在體外試驗發(fā)現(xiàn)抗菌肽Sublancin對耐甲氧西林金黃色葡萄球菌ATCC43300具有一定抑菌作用的基礎上,我們構建了耐甲氧西林金黃色葡萄球菌ATCC43300感染小鼠模型,腹腔注射2.0 mg/kg體重的Sublancin能夠降低小鼠腹腔沖洗液中金黃色葡萄球菌的數(shù)量,感染后24 h,Sublancin提高了小鼠腹腔沖洗液中TNF-α、IL-6和MCP-1的含量,而感染后72 h,Sublancin治療組小鼠腹腔沖洗液中TNF-α、IL-6和MCP-1的含量降低。(4)試驗四通過構建Caco-2細胞模型,研究Sublancin經Caco-2細胞的轉運特征,通過Cy7熒光標記Sublancin,利用活體成像近紅外熒光掃描技術,研究口服Sublancin在小鼠體內的代謝過程。在Caco-2細胞模型中,Sublancin的表觀滲透系數(shù)(Papp)小于10×10-6cm·s-1，，表明Sublancin的吸收特性較差。單次灌胃熒光染料Cy7標記的Sublancin后12 h在心、肝、脾、肺和腎組織中均沒有檢測到Cy7標記Sublancin的熒光信號,表明單次灌胃Sublancin在12h內可能未被胃腸道吸收。與游離的熒光染料Cy7相比,Cy7標記的Sublancin在小鼠腸道中停留時間更長。綜上所述,本研究證實,抗菌肽Sublancin除了具有直接的抑菌作用外,能夠通過調節(jié)小鼠天然免疫增強機體的抗細菌感染能力,開發(fā)兼?zhèn)湟志饔煤兔庖哒{節(jié)功能的抗菌肽進行抗感染治療是應對細菌耐藥性的新舉措。
[Abstract]:Four experiments to investigate the therapeutic effect of antibacterial peptide Sublancin on broiler necrotic enteritis and its effect on natural immune mice. (1) found a test in vitro antibacterial peptide Sublancin has certain inhibitory effect on Clostridium perfringens CVCC2027, a total of 252 1 day old AA broiler chickens. Were randomly divided into six treatments: normal control, Clostridium perfringens infection control, 3 Sublancin water treatment group (2.88,5.76 and 11.52 mg/L) and lincomycin drinking water treatment group (75 mg/L), the test period was 28 d. of Clostridium perfringens in d] 5-21 on the broiler challenged after 7 d Sublancin or forest clindamycine in drinking water treatment. The results showed that Sublancin alleviated the injury of intestinal villi of Clostridium perfringens infection caused by the decreased ileal proinflammatory cytokine IL-1 beta, expression of IL-6 and TNF- alpha (P0.05 ), can be reached with the forest treatment effect of kanamycin similar. (2) to test two macrophage cell line RAW264.7 as an in vitro model, activation and molecular mechanism of Sublancin on macrophages, and mouse peritoneal macrophages as experimental results validated primary cell macrophage cell lines obtained. The results showed that Sublancin significantly promoted RAW264.7 and mouse peritoneal macrophages IL-1 beta, IL-6, TNF- alpha and NO (P0.05), and the upregulation of chemokine (IL-8 and MCP-1) and costimulatory molecules (B7-1 and B7-2) gene expression (P0.05).Sublancin significantly enhanced the phagocytic function of macrophage and against methicillin-resistant Staphylococcus aureus sterilization ability, MAPK and NF- B signaling pathway involved in the activation of Sublancin on macrophages. (3) experiment three respectively in normal mice, cyclophosphamide immunosuppression and methicillin resistant Staphylococcus aureus susceptible mice The infected mice as a model research on the influence of Sublancin on natural immune mice, Sublancin mice revealed enhancement mechanism against Staphylococcus aureus infection. The results showed that normal mice fed with Sublancin, improve the phagocytic function of mouse peritoneal macrophages (P0.05), and the significant increase in mouse peritoneal macrophages IL-1 expression of IL-6 and beta. The TNF- alpha gene (P0.05). In immunosuppressed mice, Sublancin alleviated the inhibitory effect of cyclophosphamide on the phagocytic activity of macrophages in mice, alleviate the cyclophosphamide caused red blood cells in the peripheral blood hemoglobin, decrease of leukocyte and platelet content. Compared with the model group, cyclophosphamide, antibacterial peptide Sublancin (4 and 8 mg/kg body weight) after treatment, significantly up-regulated the expression of IL-2,1L-4 and IL-6 in the spleen of mouse gene (P0.05). It is found that the antibacterial peptide Sublancin on methicillin resistant West in vitro The forest based Staphylococcus aureus ATCC43300 has certain antibacterial effect, we constructed ATCC43300 in methicillin-resistant Staphylococcus aureus infection in mice by intraperitoneal injection of 2 mg/kg body weight of Sublancin can reduce the number of wash liquid in mice of Staphylococcus aureus, 24 h after infection, Sublancin improves the washing liquid by TNF- mice, the contents of IL-6 and MCP-1, and 72 h after infection, Sublancin treated mice peritoneal lavage fluid TNF- alpha, decrease the content of IL-6 and MCP-1. (4) test four through the construction of Caco-2 cell model, the study of Sublancin transport characteristics of Caco-2 cells by Cy7, fluorescence labeled Sublancin, using near infrared imaging fluorescence scan technology, study of oral Sublancin in metabolism of mice. In Caco-2 cell model, the apparent permeability of Sublancin (Papp) is less than 10 * 10-6cm, s-1, Sublanci show The poor absorption characteristics of n. Single dose Cy7 fluorescent dye labeled Sublancin after 12 h in heart, liver, spleen, lung and kidney tissues were not to Cy7 fluorescence labeled Sublancin detection showed that a single intragastric administration of Sublancin in 12h may not be absorbed in the gastrointestinal tract. Compared with free fluorescent dye Cy7, Cy7 labeled Sublancin to stay longer in the intestines of mice. In conclusion, this study demonstrated that the antibacterial peptide Sublancin has direct inhibitory effect, can enhance the body's ability to resist bacterial infection in mice by regulating innate immunity, regulating function and development of antibacterial peptide inhibitory effect and immune to anti infective therapy is a new move in response to bacterial resistance.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.31

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