本文關(guān)鍵詞： 抗菌肽Sublancin 產(chǎn)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(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：本論文通過(guò)四個(gè)試驗(yàn),探討了抗菌肽Sublancin對(duì)肉雞壞死性腸炎的治療效果及其對(duì)小鼠天然免疫的影響。(1)試驗(yàn)一在體外試驗(yàn)發(fā)現(xiàn)抗菌肽Sublancin對(duì)產(chǎn)氣莢膜梭菌CVCC2027具有一定抑菌作用的基礎(chǔ)上,選取252只1日齡愛拔益加肉公雞,隨機(jī)分為六個(gè)處理,分別為正常對(duì)照、產(chǎn)氣莢膜梭菌攻毒對(duì)照、3個(gè)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(yàn)期為28 d。在d]5-21對(duì)肉雞進(jìn)行產(chǎn)氣莢膜梭菌經(jīng)口攻毒,之后進(jìn)行持續(xù)7 d的Sublancin或林可霉素飲水治療。結(jié)果表明,Sublancin緩解了產(chǎn)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(xì)胞因子IL-1β、IL-6和TNF-α的表達(dá)量(P0.05),可達(dá)到與林可霉素相似的治療效果。(2)試驗(yàn)二以巨噬細(xì)胞系RAW264.7為體外模型,研究Sublancin對(duì)巨噬細(xì)胞的活化作用及分子機(jī)制,并用小鼠腹腔巨噬細(xì)胞作為原代細(xì)胞驗(yàn)證巨噬細(xì)胞系得到的試驗(yàn)結(jié)果。結(jié)果表明,Sublancin顯著促進(jìn)了 RAW264.7和小鼠腹腔巨噬細(xì)胞IL-1β、IL-6、TNF-α和NO的產(chǎn)生(P0.05),且上調(diào)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(dá)(P0.05)。Sublancin顯著增強(qiáng)了巨噬細(xì)胞的吞噬功能和對(duì)耐甲氧西林金黃色葡萄球菌的殺菌能力,MAPK和NF-κB信號(hào)通路參與了 Sublancin對(duì)巨噬細(xì)胞的活化過(guò)程。(3)試驗(yàn)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(duì)小鼠天然免疫的影響,揭示Sublancin增強(qiáng)小鼠抗金黃色葡萄球菌感染能力的機(jī)理。試驗(yàn)結(jié)果表明,正常小鼠中,灌服Sublancin提高了小鼠腹腔巨噬細(xì)胞的吞噬功能(P0.05),并顯著上調(diào)了小鼠腹腔巨噬細(xì)胞IL-1β、IL-6和TNF-α的基因表達(dá)(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(duì)小鼠巨噬細(xì)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(xì)胞、血紅蛋白、白細(xì)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(diào)了小鼠脾臟中IL-2、1L-4和IL-6的基因表達(dá)(P0.05)。在體外試驗(yàn)發(fā)現(xiàn)抗菌肽Sublancin對(duì)耐甲氧西林金黃色葡萄球菌ATCC43300具有一定抑菌作用的基礎(chǔ)上,我們構(gòu)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(yàn)四通過(guò)構(gòu)建Caco-2細(xì)胞模型,研究Sublancin經(jīng)Caco-2細(xì)胞的轉(zhuǎn)運(yùn)特征,通過(guò)Cy7熒光標(biāo)記Sublancin,利用活體成像近紅外熒光掃描技術(shù),研究口服Sublancin在小鼠體內(nèi)的代謝過(guò)程。在Caco-2細(xì)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(biāo)記的Sublancin后12 h在心、肝、脾、肺和腎組織中均沒有檢測(cè)到Cy7標(biāo)記Sublancin的熒光信號(hào),表明單次灌胃Sublancin在12h內(nèi)可能未被胃腸道吸收。與游離的熒光染料Cy7相比,Cy7標(biāo)記的Sublancin在小鼠腸道中停留時(shí)間更長(zhǎng)。綜上所述,本研究證實(shí),抗菌肽Sublancin除了具有直接的抑菌作用外,能夠通過(guò)調(diào)節(jié)小鼠天然免疫增強(qiáng)機(jī)體的抗細(xì)菌感染能力,開發(fā)兼?zhèn)湟志饔煤兔庖哒{(diào)節(jié)功能的抗菌肽進(jìn)行抗感染治療是應(yīng)對(duì)細(xì)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(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.31

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本文編號(hào)：1444892