牛分枝杆菌和BCG诱导树突状细胞分泌细胞因子的研究

发布时间：2018-03-04 12:17

本文选题：牛结核　切入点：卡介苗　出处：《华中农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：结核病是由结核分枝杆菌复合群引起的一种慢性消耗性的人畜共患传染病。据估计,全球大约有1/3的人感染了结核,但其中只有不到10%的人发病,这主要是由于结核分枝杆菌的感染激活了机体的免疫反应,限制了细菌的感染,最终维持了一种宿主与细菌共存的平衡状态。牛结核病是由牛分枝杆菌引起的一种慢性消耗性人兽共患传染病,可以引起包括人、家畜以及野生动物发病的疾病,被列为国际动物卫生组织(OIE)必须通报疫病之列。全球每年因结核病导致畜牧业产生的直接经济损失约为30亿美元。据统计,全世界约10%以上的人结核病是由牛分枝杆菌引起的。牛结核病给畜牧业的发展和人类健康带来重大影响,也同时成为世界性的公共卫生问题。卡介苗(BCG)是牛分枝杆菌在1908年到1921年间传了230代之后,丢失了很多区域得到的减毒株。卡介苗是目前唯一商业化应用的结核病疫苗,虽有不足,但它确实能够诱导较强的免疫保护反应。同时用卡介苗和牛分枝杆菌作为模式菌有利于发现结核的致病机制。并且,由于结核分枝杆菌与牛分枝杆菌在基因组序列上有99.95%的同源性,与结核分枝杆菌相比,牛分枝杆菌没有任何独特的基因。因此,利用BCG和毒力型牛分枝杆菌得出的发现,也有助于对结核分枝杆菌致病机制的理解。细胞因子及趋化因子在抵抗结核感染中发挥着极其重要的免疫调节作用。细胞因子能够广泛地调控机体免疫应答和造血功能,并参与炎症损伤等病理过程。趋化因子可以刺激白细胞的趋化性,吸引中性粒细胞、单核/巨噬细胞等炎性细胞移动到炎症灶,并增强炎性细胞的吞噬杀伤作用,促进它们释放炎性介质,直接参与炎症过程。本研究利用牛分枝杆菌和卡介苗感染树突状细胞,感染比为10：1,之后依次收集了感染后6h,12h和24h的培养上清,应用酶联免疫吸附试验(ELISA)测定了8种细胞因子及趋化因子的含量；通过阻断树突状细胞表面受体TLR2和TLR4以及NF-κB,检测其对细胞因子及趋化因子分泌量的影响；利用荧光定量PCR和Western blot检测了TLR/NF-κB信号通路关键分子的相对表达量。最后,本研究分析了牛分枝杆菌和BCG差异性诱导树突状细胞分泌细胞因子及趋化因子的产生及其调控通路,主要结果如下： 1.BCG与牛分枝杆菌诱导树突状细胞分泌细胞因子及趋化因子的差异收集感染后6h,12h和24h的细胞培养上清。用ELISA检测试剂盒检测了8种细胞因子及趋化因子的分泌量,包括IL-1β, IL-4, IL-6, IL-12, IL-23, TNF-a, RANTES以及MCP-1。结果显示,在未感染的DC培养上清中,IL-4的量很低,只有31.2±0.2pg/mL。感染了BCG或M. bovis后,IL-4的量会有一个缓慢的上升,但总量仍然很低。暗示在抵抗结核感染中,体液免疫的作用不是主要的。而其它的细胞因子及趋化因子(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,都有一个非常明显地升高。并且相对于M. bovis, BCG诱导了更高浓度的IL-6, IL-12, TNF-α, RANTES和MCP-1。而M. bovis则诱导了更高浓度的IL-1β和IL-23,说明M. bovis趋向于诱导炎性反应。 2.荧光定量PCR检测TLR/NF-κB信号通路关键分子的相对表达量检测了5个关键的信号通路相关基因TLR2, TLR4, Myd88, NF-κB p50和p65业基在BCG与牛分枝杆菌感染的树突状细胞中的转录差异。结果显示,BCG感染组的TLR2, TLR4, Myd88, NF-κB p50和p65亚基的转录量要高于牛分枝杆菌感染组。 3. Western blot检测NF-κB通路相关蛋白的表达 Western blot结果显示,BCG感染组的磷酸化NF-κB p65亚基的表达量明显高于M. bovis感染组；而M. bovis感染组的NF-κB的抑制业基I-κB的量则明显高于BCG感染组。这说明BCG感染组的NF-κB活化程度明显高于M bovis感染组。在使用NF-κB的抑制剂PDTC后,BCG或M. bovis感染组的磷酸化NF-κB p65亚基的量下降了,说明PDTC抑制了NF-κB的活化。 4.阻断树突状细胞表面受体TLR2、TLR4以及NF-κB后,对细胞因子及趋化因子分泌量的影响利用抗体阻断TLR2后,无论是BCG感染组还是M. bovis感染组,IL-1β,IL-6, MCP-1和RANTES的量明显被抑制了。TLR4抗体并没有此抑制效应。与此同时BCG感染组和M. bovis感染组的IL-1β, IL-6, MCP-1和RANTES的分泌可以被PDTC阻断。说明IL-1β, IL-6, MCP-1和RANTES的分泌是受TLR2/NF-κB信号通路调控的。
[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.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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