抗菌肽Sublancin对肉鸡坏死性肠炎和小鼠天然免疫的影响

发布时间：2018-01-19 16:10

本文关键词： 抗菌肽Sublancin 产气荚膜梭菌耐甲氧西林金黄色葡萄球菌天然免疫小鼠　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：本论文通过四个试验,探讨了抗菌肽Sublancin对肉鸡坏死性肠炎的治疗效果及其对小鼠天然免疫的影响。(1)试验一在体外试验发现抗菌肽Sublancin对产气荚膜梭菌CVCC2027具有一定抑菌作用的基础上,选取252只1日龄爱拔益加肉公鸡,随机分为六个处理,分别为正常对照、产气荚膜梭菌攻毒对照、3个Sublancin饮水治疗组(2.88、5.76和11.52 mg/L)和林可霉素饮水治疗组(75 mg/L),试验期为28 d。在d]5-21对肉鸡进行产气荚膜梭菌经口攻毒,之后进行持续7 d的Sublancin或林可霉素饮水治疗。结果表明,Sublancin缓解了产气荚膜梭菌攻毒造成的肠道绒毛损伤,降低了回肠促炎性细胞因子IL-1β、IL-6和TNF-α的表达量(P0.05),可达到与林可霉素相似的治疗效果。(2)试验二以巨噬细胞系RAW264.7为体外模型,研究Sublancin对巨噬细胞的活化作用及分子机制,并用小鼠腹腔巨噬细胞作为原代细胞验证巨噬细胞系得到的试验结果。结果表明,Sublancin显著促进了 RAW264.7和小鼠腹腔巨噬细胞IL-1β、IL-6、TNF-α和NO的产生(P0.05),且上调了趋化因子(IL-8和MCP-1)和共刺激分子(B7-1和B7-2)的基因表达(P0.05)。Sublancin显著增强了巨噬细胞的吞噬功能和对耐甲氧西林金黄色葡萄球菌的杀菌能力,MAPK和NF-κB信号通路参与了 Sublancin对巨噬细胞的活化过程。(3)试验三分别以正常小鼠、环磷酰胺免疫抑制小鼠和耐甲氧西林金黄色葡萄球菌感染小鼠为模型,研究Sublancin对小鼠天然免疫的影响,揭示Sublancin增强小鼠抗金黄色葡萄球菌感染能力的机理。试验结果表明,正常小鼠中,灌服Sublancin提高了小鼠腹腔巨噬细胞的吞噬功能(P0.05),并显著上调了小鼠腹腔巨噬细胞IL-1β、IL-6和TNF-α的基因表达(P0.05)。在环磷酰胺免疫抑制小鼠中,Sublancin缓解了环磷酰胺对小鼠巨噬细胞吞噬活性的抑制作用,缓解了环磷酰胺造成外周血中红细胞、血红蛋白、白细胞和血小板含量的降低。与环磷酰胺模型组相比,抗菌肽Sublancin(4.0和8.0 mg/kg体重)治疗后,显著上调了小鼠脾脏中IL-2、1L-4和IL-6的基因表达(P0.05)。在体外试验发现抗菌肽Sublancin对耐甲氧西林金黄色葡萄球菌ATCC43300具有一定抑菌作用的基础上,我们构建了耐甲氧西林金黄色葡萄球菌ATCC43300感染小鼠模型,腹腔注射2.0 mg/kg体重的Sublancin能够降低小鼠腹腔冲洗液中金黄色葡萄球菌的数量,感染后24 h,Sublancin提高了小鼠腹腔冲洗液中TNF-α、IL-6和MCP-1的含量,而感染后72 h,Sublancin治疗组小鼠腹腔冲洗液中TNF-α、IL-6和MCP-1的含量降低。(4)试验四通过构建Caco-2细胞模型,研究Sublancin经Caco-2细胞的转运特征,通过Cy7荧光标记Sublancin,利用活体成像近红外荧光扫描技术,研究口服Sublancin在小鼠体内的代谢过程。在Caco-2细胞模型中,Sublancin的表观渗透系数(Papp)小于10×10-6cm·s-1，，表明Sublancin的吸收特性较差。单次灌胃荧光染料Cy7标记的Sublancin后12 h在心、肝、脾、肺和肾组织中均没有检测到Cy7标记Sublancin的荧光信号,表明单次灌胃Sublancin在12h内可能未被胃肠道吸收。与游离的荧光染料Cy7相比,Cy7标记的Sublancin在小鼠肠道中停留时间更长。综上所述,本研究证实,抗菌肽Sublancin除了具有直接的抑菌作用外,能够通过调节小鼠天然免疫增强机体的抗细菌感染能力,开发兼备抑菌作用和免疫调节功能的抗菌肽进行抗感染治疗是应对细菌耐药性的新举措。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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