耻垢分枝杆菌动物模型的构建及其与宿主免疫系统的相互作用

发布时间：2018-02-25 06:16

本文关键词： 耻垢分枝杆菌动物模型脂阿拉伯甘露聚糖细胞因子　出处：《大连医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：结核病（Tuberclosis）是由结核分枝杆菌引起的一种严重的呼吸道传染病。结核病引起的后果对社会来说是巨大的。现在全世界三分之一的人口（约20亿人）已经感染了结核分枝杆菌，结核病占全球疾病的2.5%。尽管比起其他感染性疾病结核病的发病率有一个稳固的下降，但是除非有重大的研究结果出现，结核病持续较高死亡率的情况将持续到2020年。艾滋病和其合并形成了一个致死性的疾病组合，互相能加速发病的进程。这样的情况说明，结核病的发病与否与宿主的免疫系统有着非常密切的联系，因此结核病也成为世界关心的健康问题。[1,2,3] 分枝杆菌属包括大约100多种菌，其中包括能引起结核病的致病性分枝杆菌，如结核分枝杆菌；同样还包括非致病性的分枝杆菌，如耻垢分枝杆菌。耻垢分枝杆菌是一种与结核分枝杆菌具有同源性、快速生长、少量接种对豚鼠、大鼠、小鼠无致病性的分枝杆菌[4,5]。其细胞壁表面的有效抗原成分，如脂阿拉伯甘露糖（LAM）、脂蛋白（LP）及全细胞溶解物（Whole Cell Lysate,WCL），是与宿主免疫系统互相作用的分子[8,9]，这些分子是安全、有效的免疫调节剂，能有效地促进免疫系统的Th1应答[9]。在阐明结核病发病机制、研发疫苗等实验研究中，动物实验是最有效和不可缺少的方法[13,17]。本实验优化了构建耻垢分枝杆菌小鼠模型的方法，不但对小鼠的外部伤害减少的最低程度，同时也更真实地模拟了人类感染结核分枝杆菌的方式，以此来探讨高浓度（5×107CFU/day）耻垢分枝杆菌构建小鼠模型的可行性。本实验的目的： (1)本实验利用高浓度（5×107CFU/day）的非致病性耻垢分枝杆菌构建小鼠模型，以微生物气溶胶气雾攻击方式使小鼠肺部产生类结核状病变，来探讨利用此种方法、此种条件下，建模的可行性； (2)探讨高浓度（5×107CFU/day）的非致病性耻垢分枝杆菌在持续感染的情况下使小鼠肺部产生病理损伤的原因； (3)探讨耻垢分枝杆菌细胞壁表面成分脂阿拉伯甘露糖（LAM）对小鼠免疫系统的刺激和调节作用； (4)探讨耻垢分枝杆菌全细胞溶解物（WCL）对小鼠免疫系统的刺激和调节作用。本实验的主要方法： (1)选择近交系动物中对分枝杆菌最敏感的的C57BL小鼠，周龄为4-8周的雄性小鼠； (2)利用加入0.05%的Tween80LB液体培养基培养野生型耻垢分枝杆菌M.Smegmatis mc2155菌株； (3)利用M.Smegmatis mc2155的生长曲线与OD589测定菌液的浓度； (4)提取野生型耻垢分枝杆菌M.Smegmatis mc2155细胞壁表面物质脂阿拉伯甘露糖（LAM）； (5) M.smegmatis mc2155WCL（Whole Cell Lysate，全细胞溶解物）的制备：菌体细胞与细胞裂解液37℃孵育10min，菌液进行超声破碎，功率200W，破碎30s，间歇30s；破碎20-30个循环； (6)将实验动物C57BL小鼠随机分成5组：空白组，S组，LS组， WS1组，WS2组。各组动物分笼饲养，自由饮食饮水，饲养条件保持恒温恒湿，并定期通风消毒。 (7)小鼠的处理：各组小鼠按如下方式制备感染物，利用微生物气溶胶发生器制备感染物的气溶胶悬液，以气雾攻击方式感染小鼠。S组：5×107CFU野生型M.smegmatis MC2155；LS组：30μg/day LAM，预处理两天，第三天开始5×107CFU野生型M.smegmatis MC2155处理；WS1组：全细胞溶解物：野生型M.smegmatis MC2155=1：1混合物；WS2组：全细胞溶解物：野生型M.smegmatis MC2155=9：1混合物。 (8)分别在第7、14、21、28天，各组随机选择3只小鼠：眼球取小鼠外周血，分离血清；检测血清中IFN-γ、IL-6、IL-10、GM-CSF四中细胞因子的水平；取肺部组织，一部分做病理切片观察病理变化，另一部分用来检测肺部组织中活菌的数量。本实验的结论： (1)用气雾攻击方式，以5×107CFU/day的浓度构建耻垢分枝杆菌C57BL小鼠模型是可行的； (2)高浓度（5×107CFU/day）的耻垢分枝杆菌持续感染而使小鼠产生肺部病理损伤的原因是Th1/Th2反应失衡； (3)耻垢分枝杆菌细胞壁成分LAM能激活Th1反应具有免疫调节作用； (4) WCL与少量活菌混合后的免疫调节作用强于单独使用LAM，为研发新的疫苗奠定基础。
[Abstract]:Tuberculosis (Tuberclosis) is a serious respiratory infectious disease caused by Mycobacterium tuberculosis. The consequences are enormous for society. Now 1/3 of the world's population (about 2 billion people) has been infected with Mycobacterium tuberculosis, TB disease accounted for the global 2.5%. although the incidence of tuberculosis than other infectious diseases there is a steady decline rate, but unless there are major research results, sustained high mortality rate of tuberculosis and AIDS will continue until 2020. The merger formed a lethal disease group, each other can accelerate the onset of the process. Such a situation, whether the incidence of tuberculosis and the host immune system there is a very close relationship, therefore, TB has also become.[1,2,3] health problems in the world concern
Mycobacteria include about 100 kinds of bacteria, including Mycobacterium tuberculosis can cause illness, such as Mycobacterium tuberculosis; also includes non pathogenic mycobacteria, such as Mycobacterium smegmatis. Mycobacterium smegmatis is a Mycobacterium tuberculosis and has homology, the rapid growth of a small amount of inoculation of guinea pigs. Rat, [4,5]. mice of Mycobacterium non pathogenic antigen component of the cell wall surface effectively, such as Arabia (LAM), mannose lipid lipoprotein (LP) and whole cell lysate (Whole Cell, Lysate, WCL, [8,9]) is a molecular and host immune system interact with each other, these molecules are safe, immune effective regulator, can effectively promote the Th1 response of the immune system [9].
In the pathogenesis of tuberculosis, experimental study on developing vaccines in animal experiments, [13,17]. method is the most effective and indispensable to the optimum method of constructing Mycobacterium smegmatis mouse model, not only the lowest degree of mice external damage reduction, but also more realistic simulation of human infection with Mycobacterium tuberculosis, in order to to explore the high concentration (5 * 107CFU/day) the feasibility to establish an experimental model of Mycobacterium smegmatis.
The purpose of this experiment is:
(1) in this experiment, we established a mouse model with high concentration (5 x 107CFU/day) of non pathogenic Mycobacterium tuberculosis, and made the pulmonary tuberculosis like lesion in mice lungs by microbiological aerosol attack.
(2) to investigate the causes of pathological damage in the lungs of the non pathogenic Mycobacterium tumefaciens with high concentration (5 * 107CFU/day) in the case of persistent infection.
(3) to explore the stimulation and regulation effect of Arabia mannose (LAM) on the immune system of mice with the surface of Mycobacterium tumefaciens cell wall.
(4) to investigate the effect of WCL on the immune system of mice.
The main methods of this experiment are:
(1) the C57BL mice most sensitive to mycobacteria in inbred animals were selected, and the male mice of the week age were 4-8 weeks old.
(2) the M.Smegmatis mc2155 strain of wild Mycobacterium tumefaciens was cultivated by adding 0.05% Tween80LB liquid medium;
(3) the growth curve of M.Smegmatis mc2155 and OD589 were used to determine the concentration of the bacteria.
(4) extract Arabia mannose (LAM) on the surface of M.Smegmatis mc2155 cell wall of Mycobacterium tumefaciens;
(5) the preparation of M.smegmatis mc2155WCL (Whole Cell Lysate, total cell lysate): the cell and cell lysate were incubated at 37 degrees, 10min, the bacteria solution was ultrasonically broken, power 200W, broken 30s, intermittent 30s, and broken 20-30 cycles.
(6) the experimental animals C57BL mice were randomly divided into 5 groups: blank group, S group, LS group, WS1 group and WS2 group. All animals were divided into cage cage feeding, free diet drinking, feeding conditions kept constant temperature and humidity, and regularly ventilated and sterilized.
(7) mice: the mice were prepared according to the following infection, the use of microbial aerosol aerosol generator preparation of infected matter suspension infected mice in.S group aerosol attack: 5 * 107CFU wild type M.smegmatis MC2155; group LS: 30 g/day LAM pretreatment for two days, third days 5 * 107CFU wild type M.smegmatis MC2155 treatment; group WS1: whole cell lysate of wild-type M.smegmatis MC2155=1:1 mixture; WS2 group: whole cell lysate of wild-type M.smegmatis MC2155=9:1 mixture.
(8) after 7,14,21,28 days, each group randomly selected 3 mice: the eyeball in peripheral blood, separation of serum; the serum IFN- gamma, IL-6, IL-10, GM-CSF four cytokines; take lung tissue, as a part of pathological sections to observe the pathological changes, the other part of the used quantity of live bacteria the detection of lung tissue.
The conclusion of this experiment is:
(1) it is feasible to construct a C57BL mouse model of Mycobacterium foul with the concentration of air mist and the concentration of 5 * 107CFU/day.
(2) the high concentration (5 * 107CFU/day) of Mycobacterium tumefaciens continued to infect, and the cause of lung pathological damage in mice was the imbalance of Th1/Th2 reaction.
(3) the cell wall component LAM of Mycobacterium foul can activate the Th1 reaction with immunomodulatory effect.
(4) the immunomodulatory effect of WCL and a small amount of living bacteria is stronger than the use of LAM alone, which lays the foundation for the development of a new vaccine.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

【共引文献】