本文選題：牛結(jié)核　切入點(diǎn)：卡介苗　出處：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：結(jié)核病是由結(jié)核分枝桿菌復(fù)合群引起的一種慢性消耗性的人畜共患傳染病。據(jù)估計(jì),全球大約有1/3的人感染了結(jié)核,但其中只有不到10%的人發(fā)病,這主要是由于結(jié)核分枝桿菌的感染激活了機(jī)體的免疫反應(yīng),限制了細(xì)菌的感染,最終維持了一種宿主與細(xì)菌共存的平衡狀態(tài)。 牛結(jié)核病是由牛分枝桿菌引起的一種慢性消耗性人獸共患傳染病,可以引起包括人、家畜以及野生動(dòng)物發(fā)病的疾病,被列為國際動(dòng)物衛(wèi)生組織(OIE)必須通報(bào)疫病之列。全球每年因結(jié)核病導(dǎo)致畜牧業(yè)產(chǎn)生的直接經(jīng)濟(jì)損失約為30億美元。據(jù)統(tǒng)計(jì),全世界約10%以上的人結(jié)核病是由牛分枝桿菌引起的。牛結(jié)核病給畜牧業(yè)的發(fā)展和人類健康帶來重大影響,也同時(shí)成為世界性的公共衛(wèi)生問題。 卡介苗(BCG)是牛分枝桿菌在1908年到1921年間傳了230代之后,丟失了很多區(qū)域得到的減毒株�？ń槊缡悄壳拔ㄒ簧虡I(yè)化應(yīng)用的結(jié)核病疫苗,雖有不足,但它確實(shí)能夠誘導(dǎo)較強(qiáng)的免疫保護(hù)反應(yīng)。同時(shí)用卡介苗和牛分枝桿菌作為模式菌有利于發(fā)現(xiàn)結(jié)核的致病機(jī)制。并且,由于結(jié)核分枝桿菌與牛分枝桿菌在基因組序列上有99.95%的同源性,與結(jié)核分枝桿菌相比,牛分枝桿菌沒有任何獨(dú)特的基因。因此,利用BCG和毒力型牛分枝桿菌得出的發(fā)現(xiàn),也有助于對(duì)結(jié)核分枝桿菌致病機(jī)制的理解。 細(xì)胞因子及趨化因子在抵抗結(jié)核感染中發(fā)揮著極其重要的免疫調(diào)節(jié)作用。細(xì)胞因子能夠廣泛地調(diào)控機(jī)體免疫應(yīng)答和造血功能,并參與炎癥損傷等病理過程。趨化因子可以刺激白細(xì)胞的趨化性,吸引中性粒細(xì)胞、單核/巨噬細(xì)胞等炎性細(xì)胞移動(dòng)到炎癥灶,并增強(qiáng)炎性細(xì)胞的吞噬殺傷作用,促進(jìn)它們釋放炎性介質(zhì),直接參與炎癥過程。 本研究利用牛分枝桿菌和卡介苗感染樹突狀細(xì)胞,感染比為10：1,之后依次收集了感染后6h,12h和24h的培養(yǎng)上清,應(yīng)用酶聯(lián)免疫吸附試驗(yàn)(ELISA)測(cè)定了8種細(xì)胞因子及趨化因子的含量；通過阻斷樹突狀細(xì)胞表面受體TLR2和TLR4以及NF-κB,檢測(cè)其對(duì)細(xì)胞因子及趨化因子分泌量的影響；利用熒光定量PCR和Western blot檢測(cè)了TLR/NF-κB信號(hào)通路關(guān)鍵分子的相對(duì)表達(dá)量。最后,本研究分析了牛分枝桿菌和BCG差異性誘導(dǎo)樹突狀細(xì)胞分泌細(xì)胞因子及趨化因子的產(chǎn)生及其調(diào)控通路,主要結(jié)果如下： 1.BCG與牛分枝桿菌誘導(dǎo)樹突狀細(xì)胞分泌細(xì)胞因子及趨化因子的差異 收集感染后6h,12h和24h的細(xì)胞培養(yǎng)上清。用ELISA檢測(cè)試劑盒檢測(cè)了8種細(xì)胞因子及趨化因子的分泌量,包括IL-1β, IL-4, IL-6, IL-12, IL-23, TNF-a, RANTES以及MCP-1。結(jié)果顯示,在未感染的DC培養(yǎng)上清中,IL-4的量很低,只有31.2±0.2pg/mL。感染了BCG或M. bovis后,IL-4的量會(huì)有一個(gè)緩慢的上升,但總量仍然很低。暗示在抵抗結(jié)核感染中,體液免疫的作用不是主要的。而其它的細(xì)胞因子及趨化因子(IL-1β, IL-6, IL-12, IL-23, TNF-α, RANTES和MCP-1)的量,在DCs感染BCG或M. bovis后,都有一個(gè)非常明顯地升高。并且相對(duì)于M. bovis, BCG誘導(dǎo)了更高濃度的IL-6, IL-12, TNF-α, RANTES和MCP-1。而M. bovis則誘導(dǎo)了更高濃度的IL-1β和IL-23,說明M. bovis趨向于誘導(dǎo)炎性反應(yīng)。 2.熒光定量PCR檢測(cè)TLR/NF-κB信號(hào)通路關(guān)鍵分子的相對(duì)表達(dá)量 檢測(cè)了5個(gè)關(guān)鍵的信號(hào)通路相關(guān)基因TLR2, TLR4, Myd88, NF-κB p50和p65業(yè)基在BCG與牛分枝桿菌感染的樹突狀細(xì)胞中的轉(zhuǎn)錄差異。結(jié)果顯示,BCG感染組的TLR2, TLR4, Myd88, NF-κB p50和p65亞基的轉(zhuǎn)錄量要高于牛分枝桿菌感染組。 3. Western blot檢測(cè)NF-κB通路相關(guān)蛋白的表達(dá) Western blot結(jié)果顯示,BCG感染組的磷酸化NF-κB p65亞基的表達(dá)量明顯高于M. bovis感染組；而M. bovis感染組的NF-κB的抑制業(yè)基I-κB的量則明顯高于BCG感染組。這說明BCG感染組的NF-κB活化程度明顯高于M bovis感染組。在使用NF-κB的抑制劑PDTC后,BCG或M. bovis感染組的磷酸化NF-κB p65亞基的量下降了,說明PDTC抑制了NF-κB的活化。 4.阻斷樹突狀細(xì)胞表面受體TLR2、TLR4以及NF-κB后,對(duì)細(xì)胞因子及趨化因子分泌量的影響 利用抗體阻斷TLR2后,無論是BCG感染組還是M. bovis感染組,IL-1β,IL-6, MCP-1和RANTES的量明顯被抑制了。TLR4抗體并沒有此抑制效應(yīng)。與此同時(shí)BCG感染組和M. bovis感染組的IL-1β, IL-6, MCP-1和RANTES的分泌可以被PDTC阻斷。說明IL-1β, IL-6, MCP-1和RANTES的分泌是受TLR2/NF-κB信號(hào)通路調(diào)控的。
[Abstract]:Tuberculosis is a chronic consumption of Mycobacterium tuberculosis caused by zoonotic infectious diseases. It is estimated that there are about 1/3 of the world's people are infected with tuberculosis, but only less than 10% of the cases, this is mainly due to Mycobacterium tuberculosis infection activates the body's immune response, limit bacteria the infection, ultimately maintain a host of bacteria and coexist in equilibrium.
Bovine tuberculosis is caused by Mycobacterium bovis is a kind of chronic consumption of zoonotic infectious disease can cause diseases including human, livestock and wild animal disease, was listed as the world organization for animal health (OIE) must report the disease list. Each year due to global tuberculosis resulted in direct economic losses of livestock produce about 3 billion dollars. According to statistics, the world about more than 10% of human tuberculosis is caused by Mycobacterium bovis. Bovine tuberculosis to the development of animal husbandry and human health have a significant impact, has also become a public health problem in the world.
Bacillus Calmette Guerin (BCG) is Mycobacterium bovis from 1908 to 1921 in 230 generations after the loss of many areas. The attenuated vaccine is currently the only commercial application of TB vaccine, although insufficient, but it can induce protective immune responses stronger. At the same time with BCG and Mycobacterium bovis as model bacteria to find the pathogenic mechanism of tuberculosis. And, because have 99.95% homology in the genome sequence of Mycobacterium tuberculosis and Mycobacterium bovis, compared with Mycobacterium tuberculosis, Mycobacterium bovis no unique genes. Because of this, the use of BCG and virulent strain findings can also contribute to tuberculosis understanding of the pathogenic mechanism of Mycobacterium.
Cytokines and chemokines in the fight against tuberculosis infection plays an immunoregulatory role is extremely important. Cytokines can be widely in regulation of immune response and hematopoiesis function, and participate in inflammation and other pathological process. Chemokines can stimulate leukocyte chemotaxis, attract neutrophils, monocytes / macrophages and other inflammatory disease cells moving to inflammation, inflammatory cells and enhance phagocytic killing effect, they promote the release of inflammatory mediators, directly involved in the inflammatory process.
This research use of Mycobacterium bovis BCG infection and dendritic cells, infection ratio was 10:1, followed by collection of 6h after infection, the culture supernatant of 12h and 24h, using enzyme-linked immunosorbent assay (ELISA) determination of the content of 8 kinds of cytokines and chemokines; by blocking dendritic cell surface receptor TLR2 and TLR4 and NF- K B, to detect the effects of cytokines and chemokines secretion; to detect the relative expression of TLR/NF- kappa B pathway of key molecules using fluorescence quantitative PCR and Western blot. Finally, this study analyzed the differences of Mycobacterium bovis and BCG induced by dendritic cells to secrete cytokines and chemokines the production and regulation pathway, the main results are as follows:
Difference of cytokine and chemotactic factor in dendritic cells induced by 1.BCG and Mycobacterium bovis
Collect after infection with 6h, 12h and 24h of the cell supernatant. The secretion of 8 kinds of cytokines and chemokines were detected by ELISA kit, including IL-4, IL-6, IL-1 beta, IL-12, IL-23, TNF-a, RANTES and MCP-1. showed that in uninfected DC supernatant, the amount of IL-4 very low, only 31.2 were infected with BCG or M. + 0.2pg/mL. bovis, IL-4 will have a slow rise, but the total is still very low. In the fight against tuberculosis infection suggests that humoral immune function is not essential. But other cytokines and chemokines (beta IL-1, IL-6, IL-12. IL-23, TNF- alpha, RANTES and MCP-1) the amount of DCs in M. infected with BCG or bovis, there is a significantly increased. Compared with M. and bovis, BCG induced a higher concentration of IL-6, IL-12, TNF-, MCP-1. and M. alpha, RANTES and bovis could induce a higher concentration of IL-1 beta and IL-23, M. bovis trend To induce an inflammatory response.
Detection of relative expression of key molecules of TLR/NF- kappa B signaling pathway by 2. fluorescence quantitative PCR
Detection of 5 key signaling pathways related genes TLR2, TLR4, Myd88, NF-, P50 and B transcription difference kappa p65 subunit in BCG and Mycobacterium bovis infection of dendritic cells. The results showed that BCG infection group, TLR2, TLR4, Myd88, P50 and B transcription of NF- kappa p65 subunit to high in Mycobacterium bovis infection group.
Detection of the expression of NF- kappa B pathway related proteins by 3. Western blot
Western blot showed that the expression of phosphorylated NF- kappa B subunit p65 in BCG infection group was significantly higher than that of M. bovis infection group; and the inhibition of industry based I- kappa B M. bovis infection group NF- kappa B amount was significantly higher than that of BCG infected group. This indicated that BCG infection group NF- B activation significantly bovis infection is higher than that of M group. In the use of NF- kappa B inhibitor PDTC, the phosphorylation of NF- kappa B subunit p65 BCG or M. bovis infection group decreased, indicating that PDTC inhibited the activation of NF- K B.
4. effects of blocking the surface receptor TLR2, TLR4 and NF- kappa B on the secretion of cytokines and chemokines
The use of antibodies blocking TLR2, either BCG or M. infection group, bovis infection group, IL-1 IL-6, MCP-1 beta, and RANTES significantly inhibited.TLR4 antibody and without this inhibitory effect. At the same time BCG infection group and M. infection group IL-1 beta bovis, IL-6, MCP-1 and RANTES secretion can be blocked by PDTC. IL-1 beta, IL-6, MCP-1 and RANTES are secreted by TLR2/NF- kappa B signaling pathway.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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本文編號(hào)：1565612