本文關(guān)鍵詞：過表達(dá)oTLR4轉(zhuǎn)基因綿羊抗布魯氏菌病能力的評(píng)估及其機(jī)制研究　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

  更多相關(guān)文章： 轉(zhuǎn)基因綿羊 過表達(dá)oTLR4 感染評(píng)估 炎癥反應(yīng)

【摘要】：Toll樣受體4(Toll-like receptor 4,TLR4)是一種重要的免疫識(shí)別受體,在單核巨噬細(xì)胞,DC細(xì)胞等多種細(xì)胞中廣泛表達(dá),它識(shí)別布魯氏菌在內(nèi)的革蘭氏陰性菌的LPS,引發(fā)炎癥反應(yīng),在侵入病原體的感知與清除中發(fā)揮著重要作用。過表達(dá)oTLR4(ovis aries TLR4)的轉(zhuǎn)基因綿羊在爆發(fā)布魯氏菌病的疫區(qū)表現(xiàn)出良好的抗病潛力,因此本實(shí)驗(yàn)通過人工感染實(shí)驗(yàn)來驗(yàn)證過表達(dá)oTLR4轉(zhuǎn)基因綿羊是否有更好的抗布魯氏菌病的能力。通過PCR和Southern Blot方法,對(duì)來源于3只轉(zhuǎn)基因公羊的F1代群體共計(jì)289只進(jìn)行了過表達(dá)TLR4基因的檢測(cè),共鑒定出陽性綿羊119只,外源基因的遺傳率為41.18%,轉(zhuǎn)基因陽性綿羊TLR4 mRNA能夠穩(wěn)定高表達(dá)(平均比非轉(zhuǎn)基因個(gè)體高出1.5倍)。根據(jù)遺傳背景、品種、個(gè)體發(fā)育和轉(zhuǎn)基因與否挑選20只轉(zhuǎn)基因羊和20只非轉(zhuǎn)基因羊,分成四個(gè)攻毒劑量組。利用結(jié)膜接種的方式感染布魯氏菌16M,通過28天的綜合檢測(cè)來評(píng)估過表達(dá)oTLR4轉(zhuǎn)基因綿羊在布魯氏菌病急性期的抗病表現(xiàn)。結(jié)果低劑量(10~5和10~6CFU)和高劑量組(107,108CFU)間在急性期臨床表現(xiàn)有較大的差別。低劑量組臨床癥狀表現(xiàn)不明顯,個(gè)別羊出現(xiàn)體溫升高和白細(xì)胞增多;但高劑量組,攻毒后體溫出現(xiàn)劇烈上升,運(yùn)動(dòng)量出現(xiàn)顯著下降,WBC,淋巴細(xì)胞數(shù)等相關(guān)指標(biāo)在第7天后出現(xiàn)顯著增多,其中108劑量組在第14天檢測(cè)到白細(xì)胞數(shù)和淋巴細(xì)胞數(shù)轉(zhuǎn)基因組顯著高于非轉(zhuǎn)基因組。檢測(cè)外周血單核細(xì)胞的細(xì)胞因子表達(dá)量,結(jié)果顯示低劑量組在攻毒后0.5天、1天、2天、3天、7天,14天和28天所有時(shí)間點(diǎn)均無顯著變化,而高劑量組中,炎癥因子升高,其中108組,攻毒后7天和14天轉(zhuǎn)基因組外周血單核巨噬細(xì)胞IL6、TNFα、IL1 α和IL1 β等炎癥相關(guān)細(xì)胞因子的極顯著高于非轉(zhuǎn)基組,7天時(shí)趨化因子IL8也表現(xiàn)出轉(zhuǎn)基因組的顯著升高。結(jié)合肝臟,腎臟,脾臟和淋巴結(jié)等組織的病理切片,結(jié)果顯示隨著感染劑量的增加,實(shí)驗(yàn)羊的免疫反應(yīng)逐漸增強(qiáng),且轉(zhuǎn)基因組的增強(qiáng)幅度大于非轉(zhuǎn)基因組,在高劑量攻毒時(shí)帶來了更嚴(yán)重的組織損傷。通過虎紅檢測(cè)和微量凝集實(shí)驗(yàn),主要臟器和淋巴結(jié)的細(xì)菌分離,確定各組的感染情況,結(jié)果顯示在10~5和10~6CFU攻毒劑量組,轉(zhuǎn)基因組布魯氏菌感染數(shù)(1/10)要少于非轉(zhuǎn)基因組(3/10),通過比較載菌量和感染范圍,10~5和10~6組轉(zhuǎn)基因羊感染程度顯著低于非轉(zhuǎn)基因組。而在高劑量組中,轉(zhuǎn)基因羊(8/10)布魯氏菌感染數(shù)則要高于非轉(zhuǎn)基因羊的感染數(shù)(7/10)。轉(zhuǎn)基因組的感染程度要高于非轉(zhuǎn)基因組。通過綿羊的單核巨噬細(xì)胞侵染實(shí)驗(yàn),發(fā)現(xiàn)M0I50時(shí)侵染后1h轉(zhuǎn)基因個(gè)體載菌量極顯著高于非轉(zhuǎn)基因個(gè)體,顯示過表達(dá)TLR4提高了巨噬細(xì)胞對(duì)布魯氏菌的吞噬能力。IFNg、IL6、TNFα、Nos2和IL1 β等炎癥因子表達(dá)量極顯著高于非轉(zhuǎn)基因組,說明在感染過程中過表達(dá)TLR4提高了巨噬細(xì)胞的炎癥反應(yīng)程度。本研究驗(yàn)證了在低劑量侵染時(shí),過表達(dá)oTLR4通過提高巨噬細(xì)胞吞噬能力和炎癥反應(yīng)程度增強(qiáng)了抵抗布魯氏菌病的能力,而在高劑量侵染時(shí),過表達(dá)TLR4導(dǎo)致更嚴(yán)重的組織損傷而加重了實(shí)驗(yàn)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.
【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)】：S858.26

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本文編號(hào)：1344700