不同表型NKT细胞在产单核细胞李斯特菌L型感染小鼠中免疫作用的研究

发布时间：2018-06-28 20:44

本文选题：NKT + LM　；参考：《蚌埠医学院》2014年硕士论文

【摘要】：目的：自然杀伤性T细胞（natural killer T cell，NKT细胞）是一类既表达NK细胞表面标志又表达T细胞表面标志的免疫细胞，可以识别由抗原递呈细胞表面的CD1d分子递呈的糖脂类抗原。大量研究表明NKT细胞在抗感染免疫中发挥了重要作用。产单核细胞李斯特菌（Listeria monocytogenes，LM）是一种兼性胞内寄生菌，现已成为研究胞内菌感染机制的重要模型之一。细菌在体内外各种因素的作用下会导致细胞壁缺失，成为细菌L型。本实验室前期的研究表明，与产单核细胞李斯特菌的细菌型相比，其L型在感染小鼠后能够使肝脏和脾脏中NK1.1+NKT细胞的增殖较多，表现出NK1.1+NKT细胞的数量和比例均高于细菌型感染的小鼠。本研究将进一步研究产单核细胞李斯特菌细菌型和L型感染小鼠后，其肝脏和脾脏中NKT细胞分泌IFN-γ的情况。由于NKT细胞在C57BL/6小鼠的NKT细胞上表达NK1.1，而在各系小鼠中均可以表达DX5，所以本研究将通过尾静脉感染分别建立细菌型和L型动物感染模型，使用流式细胞仪检测感染后不同时间点不同感染组中分泌IFN-γ的不同表型NKT细胞的比例及绝对值，探讨细菌L型和细菌型对各种表型的NK1.1+NKT细胞和DX5+NKT细胞的活化能力。方法： 1、运用苯唑青霉素纸片法诱导出产单核细胞李斯特菌L型，分别调整细菌型和L型产单核细胞李斯特菌的终浓度为1×106CFU/ml； 2、分别用PBS、细菌型和L型产单核细胞李斯特菌0.1ml尾静脉感染C57BL/6小鼠，建立动物感染模型，每组6只； 3、分别在感染后第0、1、3天分离小鼠的肝脏和脾脏，采用流式细胞仪检测各组肝脏和脾脏淋巴细胞中不同表型的NKT细胞分泌IFN-γ的情况。结果： 1、产单核细胞李斯特菌诱导成为L型后，由革兰阳性转为革兰阴性，并且部分细菌出现多形性。 2、在肝脏中，DX5+NKT细胞比例和绝对值在细菌型组和L型组中随着感染时间的延长持续升高，且在感染后的第1天L型组高于细菌型组。NK1.1+NKT细胞的比例和绝对值在感染后第1天升高，第3天较第1天降低，且在感染后的第1天和第3天L型组均高于细菌型组。 3、在脾脏中，DX5+NKT细胞和NK1.1+NKT细胞的比例和绝对值在细菌型感染组中随着感染时间的延长持续升高，细胞比例在感染后第1天和第3天L型组高于细菌型组，但是绝对值在感染后第1天L型组高于细菌型组，第3天细菌型组高于L型组。 4、在肝脏中，L型细菌感染后，分泌IFN-γ的DX5+NKT细胞的比例和绝对值持续升高，在感染后第1天L型组高于细菌型组。分泌IFN-γ的的CD4+DX5+NKT细胞的比例在L型感染后持续降低，而分泌IFN-γ的的CD4-DX5+NKT细胞的比例L型感染后持续升高，而绝对值在细菌型感染后持续升高，在感染后第1天L型组高于细菌型组，第3天细菌型组高于L型组。 5、肝脏中分泌IFN-γ的NK1.1+NKT细胞的比例在细菌型感染组中第1天升高，第3天降低。在感染后第1天和第3天L型组均高于细菌型组。分泌IFN-γ的CD4+NK1.1+NKT细胞的比例在感染后第1天和第3天持续降低，第3天细菌型组高于L型组，分泌IFN-γ的CD4-NK1.1+NKT细胞的比例在感染后第1天和第3天持续升高，第3天L型组高于细菌型组，CD4+NK1.1+NKT细胞的绝对值在感染后持续升高。 6、在脾脏中，L型感染后，，分泌IFN-γ的DX5+NKT细胞的比例第1天升高，第3天降低。在感染后第1天L型组高于细菌型组。分泌IFN-γ的DX5+NKT细胞的绝对值在细菌型和L型感染组中持续升高，且在感染后第1天和第3天L型组均高于细菌型组。在细菌型感染后，分泌IFN-γ的CD4+DX5+NKT细胞的比例第1天升高第3天降低，在感染后的第3天L型组高于细菌型组。分泌IFN-γ的CD4-DX5+NKT细胞的比例第1天降低第3天升高，在感染后的第3天细菌型组高于L型组。分泌IFN-γ的CD4+DX5+NKT细胞的绝对值在L型感染后持续升高，在感染后的第1天和第3天L型组均高于细菌型组。分泌IFN-γ的CD4-DX5+NKT细胞的绝对值在细菌型和L型感染后持续升高，在感染后第1天L型组高于细菌型组，第3天细菌型组高于L型组。 7、脾脏中，分泌IFN-γ的NK1.1+NKT细胞的比例和绝对值在细菌型和L型感染后持续升高，分泌IFN-γ的NK1.1+NKT细胞的比例第1天和第3天L型组均高于细菌型组。分泌IFN-γ的CD4+NK1.1+NKT细胞的比例和绝对值在细菌型和L型感染后持续升高，第1天和第3天L型组均高于细菌型组。分泌IFN-γ的CD4-NK1.1+NKT细胞的比例在细菌型和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细胞比例细菌型组高于L型组。结论： 1、产单核细胞李斯特菌及其L型感染C57BL/6小鼠后，失去细胞壁的L型，其暴露出细胞膜上的脂类能更有效的活化DX5+NKT细胞和NK1.1+NKT细胞。NK1.1+NKT细胞在感染后可能比DX5+NKT细胞更早地发挥作用。 2、产单核细胞李斯特菌L型能更多地活化DX5+NKT细胞和NK1.1+NKT细胞分泌IFN-γ。肝脏中CD4+DX5+NKT细胞和CD4+NK1.1+NKT细胞在细菌感染后表现出不同的变化趋势。DX5+NKT细胞中CD4+DX5+NKT细胞发挥的作用持续增强，而NK1.1+NKT细胞中CD4-NK1.1+NKT细胞的作用持续增强 3、脾脏中DX5+NKT细胞在细菌型感染后发挥主要作用的是CD4-DX5+NKT细胞，NK1.1+NKT细胞在细菌型感染后发挥主要作用的是CD4-NK1.1+NKT细胞。
[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.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

【参考文献】