本文選題：NKT + LM��； 參考：《蚌埠醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的： 自然殺傷性T細(xì)胞（natural killer T cell，NKT細(xì)胞）是一類既表達(dá)NK細(xì)胞表面標(biāo)志又表達(dá)T細(xì)胞表面標(biāo)志的免疫細(xì)胞，可以識(shí)別由抗原遞呈細(xì)胞表面的CD1d分子遞呈的糖脂類抗原。大量研究表明NKT細(xì)胞在抗感染免疫中發(fā)揮了重要作用。產(chǎn)單核細(xì)胞李斯特菌（Listeria monocytogenes，LM）是一種兼性胞內(nèi)寄生菌，現(xiàn)已成為研究胞內(nèi)菌感染機(jī)制的重要模型之一。細(xì)菌在體內(nèi)外各種因素的作用下會(huì)導(dǎo)致細(xì)胞壁缺失，成為細(xì)菌L型。本實(shí)驗(yàn)室前期的研究表明，與產(chǎn)單核細(xì)胞李斯特菌的細(xì)菌型相比，其L型在感染小鼠后能夠使肝臟和脾臟中NK1.1+NKT細(xì)胞的增殖較多，表現(xiàn)出NK1.1+NKT細(xì)胞的數(shù)量和比例均高于細(xì)菌型感染的小鼠。本研究將進(jìn)一步研究產(chǎn)單核細(xì)胞李斯特菌細(xì)菌型和L型感染小鼠后，其肝臟和脾臟中NKT細(xì)胞分泌IFN-γ的情況。由于NKT細(xì)胞在C57BL/6小鼠的NKT細(xì)胞上表達(dá)NK1.1，而在各系小鼠中均可以表達(dá)DX5，所以本研究將通過尾靜脈感染分別建立細(xì)菌型和L型動(dòng)物感染模型，使用流式細(xì)胞儀檢測(cè)感染后不同時(shí)間點(diǎn)不同感染組中分泌IFN-γ的不同表型NKT細(xì)胞的比例及絕對(duì)值，探討細(xì)菌L型和細(xì)菌型對(duì)各種表型的NK1.1+NKT細(xì)胞和DX5+NKT細(xì)胞的活化能力。 方法： 1、運(yùn)用苯唑青霉素紙片法誘導(dǎo)出產(chǎn)單核細(xì)胞李斯特菌L型，分別調(diào)整細(xì)菌型和L型產(chǎn)單核細(xì)胞李斯特菌的終濃度為1×106CFU/ml； 2、分別用PBS、細(xì)菌型和L型產(chǎn)單核細(xì)胞李斯特菌0.1ml尾靜脈感染C57BL/6小鼠，建立動(dòng)物感染模型，每組6只； 3、分別在感染后第0、1、3天分離小鼠的肝臟和脾臟，采用流式細(xì)胞儀檢測(cè)各組肝臟和脾臟淋巴細(xì)胞中不同表型的NKT細(xì)胞分泌IFN-γ的情況。 結(jié)果： 1、產(chǎn)單核細(xì)胞李斯特菌誘導(dǎo)成為L(zhǎng)型后，由革蘭陽性轉(zhuǎn)為革蘭陰性，并且部分細(xì)菌出現(xiàn)多形性。 2、在肝臟中，DX5+NKT細(xì)胞比例和絕對(duì)值在細(xì)菌型組和L型組中隨著感染時(shí)間的延長(zhǎng)持續(xù)升高，且在感染后的第1天L型組高于細(xì)菌型組。NK1.1+NKT細(xì)胞的比例和絕對(duì)值在感染后第1天升高，第3天較第1天降低，且在感染后的第1天和第3天L型組均高于細(xì)菌型組。 3、在脾臟中，DX5+NKT細(xì)胞和NK1.1+NKT細(xì)胞的比例和絕對(duì)值在細(xì)菌型感染組中隨著感染時(shí)間的延長(zhǎng)持續(xù)升高，細(xì)胞比例在感染后第1天和第3天L型組高于細(xì)菌型組，但是絕對(duì)值在感染后第1天L型組高于細(xì)菌型組，第3天細(xì)菌型組高于L型組。 4、在肝臟中，L型細(xì)菌感染后，分泌IFN-γ的DX5+NKT細(xì)胞的比例和絕對(duì)值持續(xù)升高，在感染后第1天L型組高于細(xì)菌型組。分泌IFN-γ的的CD4+DX5+NKT細(xì)胞的比例在L型感染后持續(xù)降低，而分泌IFN-γ的的CD4-DX5+NKT細(xì)胞的比例L型感染后持續(xù)升高，而絕對(duì)值在細(xì)菌型感染后持續(xù)升高，在感染后第1天L型組高于細(xì)菌型組，第3天細(xì)菌型組高于L型組。 5、肝臟中分泌IFN-γ的NK1.1+NKT細(xì)胞的比例在細(xì)菌型感染組中第1天升高，第3天降低。在感染后第1天和第3天L型組均高于細(xì)菌型組。分泌IFN-γ的CD4+NK1.1+NKT細(xì)胞的比例在感染后第1天和第3天持續(xù)降低，第3天細(xì)菌型組高于L型組，分泌IFN-γ的CD4-NK1.1+NKT細(xì)胞的比例在感染后第1天和第3天持續(xù)升高，第3天L型組高于細(xì)菌型組，CD4+NK1.1+NKT細(xì)胞的絕對(duì)值在感染后持續(xù)升高。 6、在脾臟中，L型感染后，，分泌IFN-γ的DX5+NKT細(xì)胞的比例第1天升高，第3天降低。在感染后第1天L型組高于細(xì)菌型組。分泌IFN-γ的DX5+NKT細(xì)胞的絕對(duì)值在細(xì)菌型和L型感染組中持續(xù)升高，且在感染后第1天和第3天L型組均高于細(xì)菌型組。在細(xì)菌型感染后，分泌IFN-γ的CD4+DX5+NKT細(xì)胞的比例第1天升高第3天降低，在感染后的第3天L型組高于細(xì)菌型組。分泌IFN-γ的CD4-DX5+NKT細(xì)胞的比例第1天降低第3天升高，在感染后的第3天細(xì)菌型組高于L型組。分泌IFN-γ的CD4+DX5+NKT細(xì)胞的絕對(duì)值在L型感染后持續(xù)升高，在感染后的第1天和第3天L型組均高于細(xì)菌型組。分泌IFN-γ的CD4-DX5+NKT細(xì)胞的絕對(duì)值在細(xì)菌型和L型感染后持續(xù)升高，在感染后第1天L型組高于細(xì)菌型組，第3天細(xì)菌型組高于L型組。 7、脾臟中，分泌IFN-γ的NK1.1+NKT細(xì)胞的比例和絕對(duì)值在細(xì)菌型和L型感染后持續(xù)升高，分泌IFN-γ的NK1.1+NKT細(xì)胞的比例第1天和第3天L型組均高于細(xì)菌型組。分泌IFN-γ的CD4+NK1.1+NKT細(xì)胞的比例和絕對(duì)值在細(xì)菌型和L型感染后持續(xù)升高，第1天和第3天L型組均高于細(xì)菌型組。分泌IFN-γ的CD4-NK1.1+NKT細(xì)胞的比例在細(xì)菌型和L型感染后第1天升高，第3天降低，在感染后的第3天分泌IFN-γ的CD4-NK1.1+NKT細(xì)胞比例細(xì)菌型組高于L型組。 結(jié)論： 1、產(chǎn)單核細(xì)胞李斯特菌及其L型感染C57BL/6小鼠后，失去細(xì)胞壁的L型，其暴露出細(xì)胞膜上的脂類能更有效的活化DX5+NKT細(xì)胞和NK1.1+NKT細(xì)胞。NK1.1+NKT細(xì)胞在感染后可能比DX5+NKT細(xì)胞更早地發(fā)揮作用。 2、產(chǎn)單核細(xì)胞李斯特菌L型能更多地活化DX5+NKT細(xì)胞和NK1.1+NKT細(xì)胞分泌IFN-γ。肝臟中CD4+DX5+NKT細(xì)胞和CD4+NK1.1+NKT細(xì)胞在細(xì)菌感染后表現(xiàn)出不同的變化趨勢(shì)。DX5+NKT細(xì)胞中CD4+DX5+NKT細(xì)胞發(fā)揮的作用持續(xù)增強(qiáng)，而NK1.1+NKT細(xì)胞中CD4-NK1.1+NKT細(xì)胞的作用持續(xù)增強(qiáng) 3、脾臟中DX5+NKT細(xì)胞在細(xì)菌型感染后發(fā)揮主要作用的是CD4-DX5+NKT細(xì)胞，NK1.1+NKT細(xì)胞在細(xì)菌型感染后發(fā)揮主要作用的是CD4-NK1.1+NKT細(xì)胞。
[Abstract]:Objective:
Natural killer T cells (natural killer T cell, NKT cells) are immune cells that express both the surface markers of NK cells and the expression of the surface of T cells, and can identify the glycolipid antigens presented by the CD1d molecules on the surface of the antigen. A large number of studies show that NKT cells play an important role in anti infection immunity. Mononuclear cells are produced. List Rand (Listeria monocytogenes, LM) is a facultative intracellular parasitic fungus. It has now become one of the important models for the study of the mechanism of intracellular bacteria infection. Bacteria can cause cell wall deletion and become bacterial L under the action of various factors in the body and outside. The L type can increase the proliferation of NK1.1+NKT cells in the liver and spleen after infection in mice, showing that the number and proportion of NK1.1+NKT cells are higher than that of the bacterial infected mice. This study will further study the secretion of IFN- gamma in the liver and the spleen of the NKT cells in the liver and the spleen of the mice infected with the monocytic monocytic bacteria and the L type infected mice. As NKT cells express NK1.1 on NKT cells of C57BL/6 mice, DX5 can be expressed in all mice, so this study will establish bacterial and L type animal infection models by tail vein infection, and use flow cytometry to detect the proportion of the different phenotype NKT cells secreting IFN- gamma in different infection groups after infection. And absolute value, to explore the activation ability of bacteria L and bacteria to NK1.1+NKT cells and DX5+NKT cells of various phenotypes.
Method:
1, the L type of monocyte produced by benzoxacillin was used to induce the production of monocytic Lester bacteria, and the final concentration of the bacterial and L monocyte producing monocyte was 1 x 106CFU/ml, respectively.
2, C57BL/6 infected mice were infected by PBS, bacterial type and L producing monocytic Lester 0.1ml tail vein, respectively, and animal infection models were established, 6 in each group.
3, the liver and spleen of mice were separated on day 0,1,3 after infection. Flow cytometry was used to detect the secretion of IFN- gamma by NKT cells of different phenotypes in the liver and spleen lymphocytes.
Result:
1, after induction of L into monocytes, Lester transformed from gram positive to gram-negative, and some of the bacteria were polymorphic.
2, in the liver, the proportion and absolute value of DX5+NKT cells increased continuously in the bacterial group and the L group, and the proportion and absolute value of.NK1.1+NKT cells in the L group were higher than those of the bacterial group at the first day after the infection, and the third days were lower than the first day, and the L group was higher in the first and third days after the infection. In the bacterial group.
3, in the spleen, the proportion and absolute value of DX5+NKT and NK1.1+NKT cells in the bacterial infection group continued to increase with the prolongation of the infection time. The cell ratio was higher than that of the bacterial group at the first and third days after infection, but the absolute value was higher than the bacterial group in the L group after the infection, and the third day bacterial group was higher than that of the L group.
4, in the liver, the proportion and absolute value of DX5+NKT cells secreting IFN- gamma continued to rise after infection with type L bacteria, and the L group was higher than the bacterial group at the first day after infection. The proportion of CD4+DX5+NKT cells secreting IFN- gamma continued to decrease after L infection, and the proportion of CD4-DX5+ NKT cells secreting IFN- gamma continued to rise after L infection, and absolutely The value continued to rise after bacterial infection. In the first day after infection, the L group was higher than that of the bacterial group, and the bacterial type group on the third day was higher than that of the L group.
5, the proportion of NK1.1+NKT cells secreting IFN- gamma in the liver increased at first days in the bacterial infection group and decreased at third days. The L group in the first and third days after infection was higher than that in the bacterial group. The proportion of CD4+NK1.1+NKT cells secreting IFN- gamma decreased continuously at first days and third days after infection, and the bacterial group was higher than the L group and secreted the CD4-NK of IFN- gamma. The proportion of 1.1+NKT cells increased continuously at first and third days after infection. The L group was higher than the bacterial group at third days, and the absolute value of CD4+NK1.1+NKT cells increased continuously after infection.
6, in the spleen, after type L infection, the proportion of DX5+NKT cells secreting IFN- gamma increased for first days and decreased in third days. The absolute value of DX5+NKT cells secreting IFN- gamma was higher in the bacterial and L type infection group, and the L type group was higher than the bacterial group at first and third days after infection. After infection, the proportion of CD4+DX5+NKT cells secreting IFN- gamma increased third days in first days, and in third days after infection, the L group was higher than that in the bacterial group. The proportion of CD4-DX5+NKT cells secreting IFN- gamma decreased by third days, and in the third day after infection, the bacterial group was higher than the L group. The absolute value of the CD4+DX5+NKT cells secreting IFN- gamma was after the infection of the L type. In the first and third days after infection, the L group was higher than the bacterial group. The absolute value of the CD4-DX5+NKT cells secreting IFN- gamma increased continuously after the bacterial and L infection. At first days after infection, the L group was higher than the bacterial group, and the bacterial group was higher than the L group at the end of the infection.
7, in the spleen, the proportion and absolute value of NK1.1+NKT cells secreting IFN- gamma increased continuously after bacterial and L type infection, and the proportion of NK1.1+NKT cells secreting IFN- gamma was higher than that in the bacterial group first and third days. The proportion and absolute value of the CD4+NK1.1+NKT cells secreting IFN- gamma continued to increase after bacterial and L type infection, first days and third. The group of day L type was higher than that of the bacterial group. The proportion of CD4-NK1.1+NKT cells secreting IFN- gamma increased at first days after bacterial and L infection, decreased at third days, and the proportion of CD4-NK1.1+NKT cells that secreted IFN- gamma in the third day after infection was higher than that in the L group.
Conclusion:
1, after the monocytic monocytic production of monocytic monocytic bacteria and their L type infected C57BL/6 mice, the L type of cell wall is lost, and the lipids on the membrane of the cell membrane can be exposed more effectively to activate DX5+NKT and NK1.1+NKT cells,.NK1.1+NKT cells may play a role earlier than DX5+NKT cells after infection.
2, Lester producing monocytic monocytic L can more activate DX5+NKT cells and NK1.1+NKT cells to secrete IFN- gamma. CD4+DX5+NKT cells and CD4+NK1.1+NKT cells in the liver show a different trend after bacterial infection. The role of CD4+DX5+NKT cells in.DX5+NKT cells in.DX5+NKT cells continues to enhance, and CD4-NK1.1+NKT cells in NK1.1+NKT cells are made. Continuous enhancement
3, the main role of DX5+NKT cells in the infection of the spleen is CD4-DX5+NKT cells, and the main role of NK1.1+NKT cells after bacterial infection is CD4-NK1.1+NKT cells.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R392

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相關(guān)期刊論文 前3條

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本文編號(hào)：2079292