NKT细胞在李斯特菌L型感染小鼠模型中的免疫应答研究

发布时间：2018-04-20 09:25

本文选题：NKT + NK　；参考：《蚌埠医学院》2012年硕士论文

【摘要】：目的：自然杀伤T细胞(Natural killer T cells, NKT细胞)是一类介导先天性免疫和获得性免疫的免疫细胞，在抗胞内菌的感染中发挥重要作用，能通过识别APC表面CD1d提呈的糖脂类抗原（脂类抗原为主）而被活化，进而发挥作用。细菌在体内外多种因素的作用下可发生细胞壁缺失，变异成为细菌L型，导致含有较多脂质成分的细胞膜外露，因此我们推想，与野生型相比细菌L型是否能更有效的活化NKT细胞？目前国内外已有许多关于活化后的NKT细胞对细菌的非特异性应答和特异性应答的研究，但关于活化后的NKT细胞对细菌L型的应答研究还很少。因此，我们选用一种兼性胞内寄生菌，即产单核细胞李斯特菌(Listeriamonocytogenes, LM)，用青霉素诱导其成为L型，并通过尾静脉注射感染C57BL/6小鼠，分别建立野生型和L型感染的动物模型，通过检测感染小鼠肝脏和脾脏中野生型和L型细菌数（Colony-Forming Units, CFU）观察小鼠免疫系统对李斯特菌L型和野生型清除能力的差别；使用流式细胞仪检测LM野生型和L型感染小鼠后不同时间点肝、脾中NKT细胞的活化比例及绝对数，探讨细菌L型对NKT细胞的活化与野生型的差别；用酶联免疫吸附法测量不同模型中IFN-γ和IL-4在血清中的含量，比较LM及其L型感染小鼠后体内细胞因子分泌量的区别。方法： 1．用青霉素诱导LM成为L型，调整野生型和L型LM的终浓度为2×105CFU/ml； 2．通过比较几种分离方法对肝脏淋巴细胞、NKT、NK细胞比例和细胞绝对数的影响，明确不同肝脏淋巴细胞分离法的适用范围；3．分别用PBS、野生型和L型LM0.2ml尾静脉注射感染C57BL/6小鼠，建立动物感染模型，每组4只；4．感染后第3天无菌分离肝脏和脾脏，检测各组肝脏和脾脏中LM野生型和L型的CFU；5．感染后第1、3天分别分离小鼠肝脏和脾脏的淋巴细胞，采用流式细胞仪检测各组小鼠肝脏和脾脏淋巴细胞中CD3+NK1.1+NKT、CD3+NK1.1+CD4+NKT、CD3-NK1.1+NK细胞的比例及绝对数；6．摘眼球获取各组小鼠的外周血，采用酶联免疫吸附法检测各组小鼠血清中IFN-γ和IL-4的含量。结果： 1．经过青霉素纸片法诱导获得L型LM，在L型培养基中形成油煎蛋样菌落，革兰染色见部分细菌由革兰阳性转变为革兰阴性，呈多形性；细胞壁染色见细胞壁缺失所致菌体内浓染；2．小鼠肝脏淋巴细胞分离方法中，肝脏的预处理会影响淋巴细胞的获取率，不放血法获得的总淋巴细胞绝对数、T细胞比例和绝对数最高；摘眼球放血结合体循环灌流法获得的总淋巴细胞比例最高；摘眼球放血法获得的CD3+NK1.1+NKT、CD3-NK1.1+NK细胞比例最高。同时，不同的Percoll分离方法也影响各种淋巴细胞的获取，非连续密度梯度法（33%+70%Percoll）获得的淋巴细胞、T细胞、CD3+NK1.1+NKT、CD3-NK1.1+NK细胞比例均高于33%组； 3．L型感染组小鼠在感染后第3天肝脏和脾脏的L型CFU数均明显低于野生型感染组； 4．在感染后的第1天和第3天，比较小鼠肝脏淋巴细胞中的CD3+NK1.1+NKT、CD3+NK1.1+CD4+NKT、CD3-NK1.1+NK细胞的比例和绝对数发现，L型感染组的上述细胞均高于野生型感染组；2个感染组的上述细胞中，除了第1天CD3+NK1.1+CD4+NKT细胞的比例外，其它细胞各项数值均高于未感染组；其中CD3+NK1.1+NKT、CD3-NK1.1+NK细胞的比例和绝对数在第3天较第1天略有下降，野生型和L型感染组CD3+NK1.1+CD4+NKT的绝对数在第1天和第3天均升高，而第3天较第1天下降，但其比例在L型组中第3天升高，野生型组在第1天下降，第3天升高。 5．比较小鼠脾脏内CD3+NK1.1+NKT和CD3-NK1.1+NK细胞比例和绝对数，发现L型组均高于野生型组，而且2个感染组均高于未感染组，，与肝脏中不同的是这2种细胞的比例和绝对数在第3天均高于第1天。与未感染组相比，2个感染组小鼠脾脏中CD3+NK1.1+CD4+NKT细胞的比例在感染后第1天时升高，第3天较未感染组低，但是其绝对数在第1和第3天均高于未感染组。L型感染组在感染第1天CD3+NK1.1+CD4+NKT细胞比例高于，但是在第3天却低于野生型组，而细胞绝对数却均高于野生型组。 6．ELISA检测感染小鼠外周血分泌的IFN-γ和IL-4的分泌情况，结果显示随着感染时间的增加，血清中IFN-γ水平不断增加，且L型组高于野生型；而随着感染时间的增加，血清中的IL-4水平先增加后降低，且L型组比野生型组更低。结论： 1．采用摘眼球放血结合心脏灌流的方法获得肝脏单个核细胞悬液，然后采用非连续密度梯度离心法分离淋巴细胞，可获得较大比例的肝脏淋巴细胞及其亚群。 2．在小鼠的肝脏和脾脏中，感染的李斯特菌L型比野生型更容易被清除。 3．李斯特菌L型能更有效的刺激CD3+NK1.1+NKT、CD3+NK1.1+CD4+NKT、CD3-NK1.1+NK细胞的增殖，这可能是细菌L型更容易被清除的原因之一。 4．肝脏作为固有免疫器官，在抗李斯特菌及其L型感染的非特异性免疫过程中，肝脏中的NKT和NK细胞在感染早期发挥作用的高峰比在脾脏更早。 5．在抗李斯特菌及其L型感染中，肝脏中活化后的CD3+NK1.1+NKT细胞可能以CD3+NK1.1+CD4+NKT亚群发挥主要作用，而脾脏中则以CD3+NK1.1+CD4-NKT为主。 6．与李斯特菌野生型相比，L型感染能诱发机体的免疫系统产生更多的IFN-γ发挥抗感染作用。
[Abstract]:Purpose :

Natural killer T cells ( NKT cells ) , a kind of immune cells mediating innate immunity and acquired immunity , play an important role in the infection of intracellular bacteria .
The activation ratio and absolute number of NKT cells in liver and spleen of LM wild - type and L - type infected mice were detected by flow cytometry .
The levels of IFN - 纬 and IL - 4 in serum were measured by enzyme - linked immunosorbent assay ( ELISA ) , and the difference of cytokine secretion was compared between LM and L - type infected mice .

Method :

1 . using penicillin to induce LM to become L - type , adjusting the final concentration of wild - type and L - type LM to 2 & # xd7 ; 105 CFU / ml ;

2 . The effects of several methods of separation on the ratio of lymphocyte , NKT , NK cells and absolute number of cells were compared .
3 . C57BL / 6 mice were infected with PBS , wild - type and L - type LM0.2 ml tail vein , and animal infection model was established .
4 . The liver and spleen were isolated after infection on Day 3 after infection , and the CFU of LM wild type and L type were detected in the liver and spleen of each group ;
5 . Lymphocytes from mouse liver and spleen were isolated on days 1 and 3 after infection . CD3 + NK1 . 1 + NKT , CD3 + NK1 . 1 + CD4 + NKT , CD3 + NK1 . 1 + NK cells and absolute numbers of CD3 + NK1 . 1 + CD4 + NKT , CD3 + NK1 . 1 + NK cells were detected by flow cytometry .
6 . The contents of IFN - 纬 and IL - 4 in serum of each group were determined by enzyme - linked immunosorbent assay ( ELISA ) .

Results :

1 . L - type LM was induced by penicillin paper method . In L - type culture medium , oil - fried egg - like colony was formed , and Gram - positive transformation of Gram - negative bacteria was found to be negative in Gram - negative .
Cell wall staining was found in the presence of cell wall deletion .
2 . In the method of mouse liver lymphocyte separation , the pretreatment of the liver could affect the rate of lymphocyte acquisition , the absolute number of total lymphocytes obtained by the non - bleeding method , the percentage of T cells and the highest absolute number ;
The proportion of total lymphocytes obtained by the method of blood circulation and blood circulation was the highest .
The ratio of CD3 + NK1 . 1 + NKT , CD3 - NK1 . 1 + NK cells was the highest , and the ratio of lymphocytes , T cells , CD3 + NK1 . 1 + NKT , CD3 - NK1 . 1 + NK cells was higher than that of 33 % .

3 . The number of L - shaped CFU in the liver and spleen of the L - type infected group was significantly lower than that in the wild type infection group on the third day after infection .

4 . The proportion and absolute number of CD3 + NK1 . 1 + NKT , CD3 + NK1 . 1 + CD4 + NKT , CD3 + NK1 . 1 + NK cells in the liver lymphocytes of mice were compared with wild type infection group on days 1 and 3 after infection .
Except for CD3 + NK1 . 1 + CD4 + NKT cells in the first day , the numbers of other cells were higher than those in the non - infected group .
The ratio and absolute number of CD3 + NK1 . 1 + NKT , CD3 - NK1 . 1 + NK cells decreased slightly on Day 1 and Day 1 , while the absolute numbers of CD3 + NK1 . 1 + CD4 + NKT in wild type and L - type infection groups increased on Day 1 and Day 3 , while the third day decreased from Day 1 , but the proportion of CD3 + NK1 . 1 + CD4 + NKT increased on Day 3 in the L - type group and the wild - type group decreased on Day 1 and increased on Day 3 .

The percentage and absolute numbers of CD3 + NK1 . 1 + NKT and CD3 - NK1 . 1 + NK cells in spleen of mice were higher than those in the non - infected group . The percentage and absolute numbers of CD3 + NK1 . 1 + CD4 + NKT cells in the spleen of the two infected groups were higher than those in the uninfected group .

6 . ELISA was used to detect the secretion of IFN - 纬 and IL - 4 in peripheral blood of infected mice . The results showed that the level of IFN - 纬 in serum increased with the increase of infection time , and the L - type group was higher than that of wild type ;
However , with the increase of infection time , the level of IL - 4 in serum decreased first , and L - type group was lower than that of wild - type group .

Conclusion :

1 . The mononuclear cell suspension of the liver was obtained by the method of exophthalmos and cardiac perfusion , then lymphocytes were separated by discontinuous density gradient centrifugation , and a large proportion of the liver lymphocytes and their sub - groups were obtained .

2 . In the liver and spleen of the mouse , the type L of the infection is more easily removed than the wild type .

3 . The proliferation of CD3 + NK1 . 1 + NKT , CD3 + NK1 . 1 + CD4 + NKT , CD3 - NK1 . 1 + NK cells could be more effectively stimulated by L - type . This could be one of the reasons for easier removal of bacterial L - type .

4 . As an innate immune organ , the peak ratio of NKT and NK cells in the liver to the early stage of infection was earlier in the early stage of infection than in the spleen .

5 . The CD3 + NK1 . 1 + NKT cells activated in the liver may play a major role in the CD3 + NK1 . 1 + CD4 + NKT sub - group and CD3 + NK1 . 1 + CD4 - NKT in the spleen .

6 . L - type infection can induce more IFN - 纬 to play an anti - infective role in the body ' s immune system than wild - type strain .

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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