过表达oTLR4转基因绵羊抗布鲁氏菌病能力的评估及其机制研究

发布时间：2017-12-28 05:13

本文关键词：过表达oTLR4转基因绵羊抗布鲁氏菌病能力的评估及其机制研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：Toll样受体4(Toll-like receptor 4,TLR4)是一种重要的免疫识别受体,在单核巨噬细胞,DC细胞等多种细胞中广泛表达,它识别布鲁氏菌在内的革兰氏阴性菌的LPS,引发炎症反应,在侵入病原体的感知与清除中发挥着重要作用。过表达oTLR4(ovis aries TLR4)的转基因绵羊在爆发布鲁氏菌病的疫区表现出良好的抗病潜力,因此本实验通过人工感染实验来验证过表达oTLR4转基因绵羊是否有更好的抗布鲁氏菌病的能力。通过PCR和Southern Blot方法,对来源于3只转基因公羊的F1代群体共计289只进行了过表达TLR4基因的检测,共鉴定出阳性绵羊119只,外源基因的遗传率为41.18%,转基因阳性绵羊TLR4 mRNA能够稳定高表达(平均比非转基因个体高出1.5倍)。根据遗传背景、品种、个体发育和转基因与否挑选20只转基因羊和20只非转基因羊,分成四个攻毒剂量组。利用结膜接种的方式感染布鲁氏菌16M,通过28天的综合检测来评估过表达oTLR4转基因绵羊在布鲁氏菌病急性期的抗病表现。结果低剂量(10~5和10~6CFU)和高剂量组(107,108CFU)间在急性期临床表现有较大的差别。低剂量组临床症状表现不明显,个别羊出现体温升高和白细胞增多;但高剂量组,攻毒后体温出现剧烈上升,运动量出现显著下降,WBC,淋巴细胞数等相关指标在第7天后出现显著增多,其中108剂量组在第14天检测到白细胞数和淋巴细胞数转基因组显著高于非转基因组。检测外周血单核细胞的细胞因子表达量,结果显示低剂量组在攻毒后0.5天、1天、2天、3天、7天,14天和28天所有时间点均无显著变化,而高剂量组中,炎症因子升高,其中108组,攻毒后7天和14天转基因组外周血单核巨噬细胞IL6、TNFα、IL1 α和IL1 β等炎症相关细胞因子的极显著高于非转基组,7天时趋化因子IL8也表现出转基因组的显著升高。结合肝脏,肾脏,脾脏和淋巴结等组织的病理切片,结果显示随着感染剂量的增加,实验羊的免疫反应逐渐增强,且转基因组的增强幅度大于非转基因组,在高剂量攻毒时带来了更严重的组织损伤。通过虎红检测和微量凝集实验,主要脏器和淋巴结的细菌分离,确定各组的感染情况,结果显示在10~5和10~6CFU攻毒剂量组,转基因组布鲁氏菌感染数(1/10)要少于非转基因组(3/10),通过比较载菌量和感染范围,10~5和10~6组转基因羊感染程度显著低于非转基因组。而在高剂量组中,转基因羊(8/10)布鲁氏菌感染数则要高于非转基因羊的感染数(7/10)。转基因组的感染程度要高于非转基因组。通过绵羊的单核巨噬细胞侵染实验,发现M0I50时侵染后1h转基因个体载菌量极显著高于非转基因个体,显示过表达TLR4提高了巨噬细胞对布鲁氏菌的吞噬能力。IFNg、IL6、TNFα、Nos2和IL1 β等炎症因子表达量极显著高于非转基因组,说明在感染过程中过表达TLR4提高了巨噬细胞的炎症反应程度。本研究验证了在低剂量侵染时,过表达oTLR4通过提高巨噬细胞吞噬能力和炎症反应程度增强了抵抗布鲁氏菌病的能力,而在高剂量侵染时,过表达TLR4导致更严重的组织损伤而加重了实验羊的感染程度。
[Abstract]:Toll like receptor 4 (Toll-like receptor 4, TLR4) is a kind of important immune recognition receptors in macrophages, extensive expression of a variety of DC cells and other cells, gram negative bacteria, it identifies Brucella LPS, trigger inflammation, plays an important role in the invasion of pathogen perception and elimination. Transgenic sheep with over expression of oTLR4 (Ovis aries TLR4) showed good resistance potential in the outbreak area of brucellosis. Therefore, we experimentally verified whether oTLR4 transgenic sheep had better ability to resist brucellosis by artificial infection test. Through the PCR and the Southern Blot method, from 3 transgenic ram F1 generation of a total of 289 was carried out to detect the expression of TLR4 gene, were identified positive 119 sheep genetic gene transgenic positive rate was 41.18%, TLR4 mRNA high expression in sheep (the average is 1.5 times higher than that of non transgenic individuals). 20 transgenic sheep and 20 non transgenic sheep were selected according to the genetic background, variety, ontogeny and GM or not, and were divided into four drug attack dose groups. The infection of Brucella 16M was infected by conjunctival inoculation. We evaluated the disease resistance of oTLR4 transgenic sheep in the acute stage of brucellosis in 28 days. Results the clinical manifestations of low dose (10~5 and 10~6CFU) and high dose group (107108CFU) were significantly different in the acute phase. The low dose group of clinical symptoms was not obvious, individual sheep appeared fever and leukocytosis; but in high dose group, after infection of severe temperature rise, the amount of exercise is significantly decreased, WBC lymphocyte number index significantly increased in seventh days, including 108 dose group in Fourteenth days and the number of lymphocytes detected the number of white blood cell transfer group was significantly higher than that of non transgenic group. The expression of cytokines in the detection of peripheral blood mononuclear cells. The results show that the low dose group of inoculation after 0.5 days, 1 days, 2 days, 3 days, 7 days, 14 days and 28 days for all time points had no significant change, and the high dose group, inflammatory cytokines, including 108 groups. After infection of 7 days and 14 days of transgenic group peripheral blood mononuclear macrophage IL6, TNF and IL1 alpha and IL1 beta and other cytokines were significantly higher than that of non transgenic group, 7 days of chemokine IL8 also showed significantly increased in transgenic. Combined with pathological sections of liver, kidney, spleen and lymph nodes, the results showed that with the increase of infection dose, the immune response of the experimental sheep increased gradually, and the amplification of the genome increased more than that of the non transferred genomes. The tiger red detection and micro agglutination test, bacterial isolation of main organs and lymph nodes, determine infection groups, results showed that the inoculation dose group in 10~5 and 10~6CFU, the number of transgenic group of Brucella infection (1/10) than non transgenic (3/10), by comparing the microbial load and the infection of 10~5 and 10~6. Group of transgenic sheep was significantly lower than that in non transgenic infection. In the high dose group, the number of Brucella infection in transgenic sheep (8/10) was higher than that of non transgenic sheep (7/10). The degree of infection in the transgenic group was higher than that of the non - transgenome. Through the mononuclear macrophage infection experiment in sheep, it was found that the infection rate of 1H transgenic individuals was significantly higher than that of non transgenic individuals when M0I50 was infected, indicating that over expression of TLR4 increased macrophage phagocytosis ability of Brucella. The expression levels of IFNg, IL6, TNF alpha, Nos2 and IL1 beta were significantly higher than those of non transferred genomes, indicating that over expression of TLR4 during infection increased the inflammatory response of macrophages. This study tested in low dose infection, overexpression of oTLR4 by improving the phagocytic capacity and the degree of inflammatory reaction and enhance the resistance of brucellosis, and in high dose infection, overexpression of TLR4 resulted in more severe tissue damage and increased the degree of experimental infection of sheep.
【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】：S858.26

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