發(fā)布時間：2018-02-14 20:18

  本文關鍵詞： 漿細胞樣樹突狀細胞 剛地弓形蟲 CD4~+T細胞過度極化　出處：《山東農業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：剛地弓形蟲是一種專性細胞內寄生蟲,感染免疫功能正常的宿主通常無癥狀,在免疫缺陷或者先天性感染的宿主中可成為致死性的機會致病原蟲。樹突狀細胞(Dendritic Cells,DCs)是機體在弓形蟲感染早期最先產生應答的固有免疫細胞之一,DCs作為免疫系統(tǒng)中最重要的抗原提呈細胞,可以活化T細胞,啟動宿主的獲得性免疫反應,是連接固有免疫和適應性免疫的橋梁。DCs是高度異質性細胞群體,包含多種亞群。由于DCs的生物學效應的發(fā)揮取決于其細胞類型和數(shù)量,因此研究DCs亞群在抗弓形蟲感染中的作用成為本領域研究的熱點。樹突狀細胞可以分為經典性樹突狀細胞(Conventional dendritic cells,cDCs)和漿細胞樣樹突狀細胞(Plcasmacytiod Dendritic Cells,pDCs)。pDCs是DCs的一個獨特亞群,在不同微環(huán)境下可以選擇性的誘導免疫應答也可以誘導免疫耐受。近來有文獻報道pDCs在弓形蟲感染時可以識別弓形蟲的抗原,將弓形蟲的抗原提呈給T細胞,以啟動適應性免疫應答。清除pDCs增加了TRL11-/-基因缺陷小鼠對弓形蟲的易感性。但是pDCs在弓形蟲感染引起的急性炎癥中對固有免疫和適應性免疫反應的影響機制仍不十分清楚,因此,本課題應用現(xiàn)代免疫學方法并結合使用pDCs條件性基因敲除小鼠,力圖闡明pDCs在抗弓形蟲感染中對固有免疫和適應性免疫細胞的調控機制。該研究有助于更好的理解pDCs在感染免疫中的作用和貢獻,為研發(fā)有效的抗弓形蟲樹突狀細胞疫苗提供新的靶點。本研究主要是通過以下幾個方面進行并且得到如下結果:1.pDCs缺失增加了小鼠對弓形蟲的易感性本研究使用BDCA2-DTR小鼠腹腔注射DT,特異性清除pDCs,隨后腹腔感染弓形蟲速殖子后,感染三天后發(fā)現(xiàn),清除pDCs的小鼠與對照組相比極大的增強了對弓形蟲的易感性,脾臟腫大,死亡率增加了45%。該結果提示pDCs在抗弓形蟲感染中起到非常重要的作用。2.pDCs缺失導致了IL-12分泌減少檢測感染小鼠血清中分泌的IL-12發(fā)現(xiàn),與對照組相比pDCs缺失小鼠IL-12p40分泌顯著性減少,IL-12p70未檢測到。該結果提示pDCs在IL-12的合成和分泌中起到調控作用。3.pDCs缺失加重了弓形蟲感染導致的Th1型細胞因子分泌檢測感染小鼠血清中分泌的Th1型細胞因子水平發(fā)現(xiàn),高分泌的IFN-γ、IL-6、TNF-α、在pDCs缺失后顯著性的增加了。該結果提示pDCs缺失引起了Th1型細胞因子風暴。4.pDCs缺失影響巨噬細胞和cDCs細胞數(shù)量檢測感染小鼠脾臟的效應細胞發(fā)現(xiàn),pDCs缺失并不影響固有免疫細胞中的中性粒細胞、炎癥性單核細胞數(shù)量。但是引起巨噬細胞、cDCs數(shù)量顯著性減少。進一步分析cDCs的兩個亞群CD8+DCs和CD11b+DCs的變化發(fā)現(xiàn),pDCs缺失主要引起了CD8+DCs的比例和數(shù)量顯著性減少。對適應性免疫細胞CD4+T、CD8+T細胞檢測發(fā)現(xiàn),弓形蟲感染導致了T細胞顯著性減少,pDCs缺失并不影響T細胞數(shù)量。該結果提示pDCs和巨噬細胞、CD8+DCs存在交叉對話。5.pDCs缺失引起CD4+T細胞過度極化對體內主要分泌IFN-γ的細胞檢測發(fā)現(xiàn),弓形蟲感染3天后,IFN-γ主要是由CD4+T細胞分泌的,pDCs缺失不影響NK細胞分泌的IFN-γ,IFN-γ陽性的CD4+T比例和數(shù)量均顯著性增加,CD8+T比例著性增加,但是細胞數(shù)量顯著性減少。該結果提示pDCs缺失導致了CD4+T細胞過度活化。綜上所述,得出如下結論:pDCs影響IL-12的分泌,在早期抗弓形蟲感染中起到保護性作用,影響固有免疫細胞中的巨噬細胞、CD8+DCs的細胞數(shù)量,影響適應性免疫細胞中CD4+T分泌IFN-γ的能力,影響Th1型細胞因子分泌。
[Abstract]:Toxoplasma gondii is an obligate intracellular parasite infection in immunocompetent hosts are usually asymptomatic, or congenital infection in immunocompromised hosts can become lethal opportunistic protozoan. Dendritic cells (Dendritic Cells DCs) is one of the body's innate immune cells in early first response to Toxoplasma infection DCs, as the most important immune system in antigen-presenting cells and can activate T cells, initiating host acquired immune responses,.DCs is a bridge between innate immunity and acquired immunity is a highly heterogeneous cell population contains multiple subsets. The biological effects of DCs depends on the cell type and quantity. The role of DCs subsets in anti Toxoplasma gondii infection has become a hot research in this field. Dendritic cells can be divided into classical dendritic cells (Conventional dendritic cell S, cDCs) and plasmacytoid dendritic cells (Plcasmacytiod Dendritic Cells, pDCs.PDCs DCs) is a unique subtype, can induce immune response selectivity can also induce immune tolerance in different micro environment. It has recently been reported in pDCs of Toxoplasma gondii infection can identify Toxoplasma antigens of Toxoplasma gondii the antigens to T cells to initiate adaptive immune responses. PDCs clearance increased susceptibility to Toxoplasma gondii TRL11-/- gene deficient mice. But pDCs in acute inflammation caused by the infection of Toxoplasma gondii in the effect and the mechanism of innate and adaptive immune response is still not very clear, therefore, the application of modern immunology method combined with the use of pDCs conditional knockout mice, to clarify pDCs in anti Toxoplasma gondii infection on innate and adaptive immune cell regulation mechanism. The study is very helpful to science The solution and the contribution of pDCs in infectionimmunity, for the development of effective anti Toxoplasma dendritic cell vaccines provide a new target. This research is mainly through the following aspects and results are as follows: 1.pDCs deletion increased susceptibility to Toxoplasma gondii in mice in this study using BDCA2-DTR mice by intraperitoneal injection of DT, specific clearance pDCs, then the abdominal infection of Toxoplasma gondii infection after three days found that the clearance of pDCs mice compared with the control group greatly enhanced the susceptibility to Toxoplasma gondii, splenomegaly, mortality increased 45%. the results of pDCs in anti Toxoplasma infection plays a very important role in.2.pDCs deletion led to IL-12 reduce the secretion of IL-12 secretion in infected mice was measured in serum showed that compared with the control group pDCs deficient mice significantly reduced the secretion of IL-12p40 and IL-12p70 were not detected. The results suggest that pDCs in IL-12 The synthesis and secretion of play a regulatory role in.3.pDCs deletion aggravated the Toxoplasma infection of Th1 type cytokines leads to the secretion of Th1 type cytokines secreted by detection of infected mice found in serum, IFN- gamma, hypersecretion of IL-6, TNF- alpha, in after deletion of pDCs was significantly increased. The results suggest that pDCs deficiency caused Th1 type cytokine storm.4.pDCs deletion effect cell number of macrophages and cDCs cells were detected in infected mice spleen showed that pDCs deletion did not affect neutrophil innate immune cells, the number of inflammatory mononuclear cells by macrophages. However, the number of cDCs significantly reduced. Changes are found in the further analysis of cDCs two subsets of CD8+DCs and CD11b+DCs, pDCs deletion is mainly caused by the CD8+DCs number and the proportion were significantly reduced. The adaptive immune cells CD4+T, CD8+T cell detection of Toxoplasma gondii infection Guide Due to the significant decrease of T cells, pDCs deletion did not affect the number of T cells. The results suggest that pDCs and CD8+DCs macrophages, the cross-talk between.5.pDCs cells that detect CD4+T cell loss caused by excessive polarization on the main body of IFN- secretion, 3 days after infection with Toxoplasma gondii, IFN- gamma is mainly secreted by CD4+T cells, deletion of pDCs does not effects of IFN- gamma NK cells, IFN- positive ratio of CD4+T and the number were significantly increased, the proportion of CD8+T increased significantly, but the number of cells significantly reduced. The results suggest that pDCs deficiency led to the excessive activation of CD4+T cells. In summary, draw the following conclusions: influence the secretion of IL-12 pDCs, in the early stage of anti Toxoplasma infection to play a protective effect, effect of innate immune cells in macrophages, the number of CD8+DCs cells and the effect of CD4+T in adaptive immune cells to secrete IFN-, effect of Th1 type cytokines Secrete.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S855.9

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