大鼠外周血中性粒细胞在较长细菌刺激时期内的生物学反应特性及其意义

发布时间：2017-12-31 12:00

本文关键词：大鼠外周血中性粒细胞在较长细菌刺激时期内的生物学反应特性及其意义　出处：《西北大学》2007年硕士论文　论文类型：学位论文

【摘要】： 中性粒细胞是一类重要的吞噬细胞，是机体非特异性免疫系统抗御病原微生物感染的重要屏障。目前普遍认为中性粒细胞的抑菌杀菌机制是通过吞噬、灭活、消化等细胞内机制实现的。其细胞内抑菌杀菌作用与呼吸爆发时NADPH氧化酶介导产生的活性氧(reactive oxygen species，ROS)及颗粒泡内蛋白水解酶直接相关，此过程在30分钟左右基本完成。这种抑菌杀菌机制的建立主要基于利用纯化的中性粒细胞进行的大量体外实验研究结果。这些研究主要观察从中性粒细胞与调理素结合的细菌或产物混合至呼吸爆发完成很短时间内相应实验指标的变化，缺乏有关反应时间长于30分钟的实验资料的报道。本文研究了较长细菌刺激时期内(约300分钟)大鼠外周血中性粒细胞的生物学反应特性及其意义。主要采用细胞耗氧、化学发光等检测方法研究系统氧耗变化，活性氧物质生成反应，用细菌培养方法研究中性粒细胞抑菌杀菌机制，采用细胞培养、染色、光学显微镜形态学方法研究测定中性粒细胞吞噬功能。在细菌与外周血混合后，血细胞(中性粒细胞)系统产生两个相互分离的ROS生成峰，即ROS峰Ⅰ和ROS峰Ⅱ。ROS峰Ⅰ在刺激后立即出现，30分钟以内消失，峰值小，伴有耗氧增强；ROS峰Ⅱ在刺激后60分钟左右出现，持续时间可达150分钟以上，峰值明显高于峰Ⅰ，不伴耗氧现象。ROS峰Ⅰ的大小与细菌浓度成正比，其峰值出现时间无明显改变；ROS峰Ⅱ随细菌浓度增加，其峰值出现时间明显提前，峰值逐渐增加。整个历时近300分钟的反应过程中，用辣根过氧化物酶未测出细胞外发光信号，表明整个过程中产生化学发光的中性粒细胞细胞膜通透性无明显变化，其发光反应主要来自细胞内生物化学反应过程。显然，，ROS峰Ⅰ和ROS峰Ⅱ具有明显不同的生物学活性，并且代表两类不同功能状态的细胞。用绿脓杆菌等进行的体外实验研究还发现，大鼠全血具有明显的抑菌杀菌作用，此作用与中性粒细胞直接相关，但与血浆成份无直接关系。全血抑菌杀菌过程可分成两个截然不同的阶段，即在ROS峰Ⅰ前期的快速抑菌杀菌期和其后的缓慢抑菌杀菌期。快速抑菌杀菌期中细菌灭活率高达90％左右，此作用在反应开始后3分钟内基本完成。此时，中性粒细胞吞噬细菌的程度并不明显，吞噬细菌中性粒细胞的比例仅占总粒细胞的5％以下，显然，此间的抑菌杀菌现象与中性粒细胞的胞外作用机制有关。在缓慢抑菌杀菌期，中性粒细胞吞噬细菌的作用随反应时间延长而明显增强，在ROS峰Ⅱ之后(反应后200-250分钟)，其吞噬率可达30％-60％。吞噬中性粒细胞的比例以及单位粒细胞吞噬细菌的数量随培养时间的延长而增加的程度与反应系统中活菌数量的减少幅度基本相似，因此，中性粒细胞的吞噬及胞内机制可能在缓慢抑菌杀菌期中起着重要的作用。综上所述，我们认为外周血中性粒细胞至少可分为两大种类，即：易被外来物质激活、吞噬和／或消化活性伴随明显耗氧的早期反应中性粒细胞和激活缓慢、吞噬和／或消化活性不伴随耗氧现象的迟缓反应中性粒细胞。在体内抗感染过程中，细胞外作用机制是中性粒细胞抑菌杀菌的重要手段。中性粒细胞吞噬及其相关的细胞内机制主要在于外来异物，例如：病源微生物，的体内清除。本文还对本研究有关中性粒细胞抑菌杀菌机理的实验结果与目前通常认为的作用机理差异的原因进行了讨论。
[Abstract]:Neutrophils are an important class of phagocytic cells, nonspecific immune system is an important barrier against pathogen infection. It is generally believed that the mechanism of antibacterial sterilization of neutrophils through phagocytosis, inactivation, digestion and other implementation mechanisms in the cell. The cell inhibitory and bactericidal effects of respiratory burst oxidase mediated NADPH guide the production of reactive oxygen species (reactive oxygen, species, ROS) and particle bubble proteolytic enzymes is directly related to the basic completion of this process in 30 minutes. The bactericidal mechanisms mainly in neutrophils by purification based on a large number of the in vitro experiments. These studies mainly from the observation of neutrophils and opsonin the combination of bacteria or product mix to complete the change of respiratory burst in a very short period of time corresponding experimental indexes, the lack of relevant experimental data the reaction time is longer than 30 minutes. Report.
This paper studies the long period of bacterial stimulation (about 300 minutes) in peripheral blood of rats of neutral granulocyte biological reaction characteristics and its significance. Mainly by cell oxygen consumption, chemiluminescence detection method to study the system of oxygen consumption changes, ROS formation reaction, culture of neutrophil bactericidal mechanisms used by bacteria cell culture, staining, microscope optical morphological method for determination of phagocytic function of neutrophils. In bacteria and peripheral blood after mixed blood cells (neutrophils) system to produce two separate ROS generation peak, ROS peak ROS peak.ROS peak I and II I immediately appear in the stimulation, disappeared, 30 within minutes a small peak, with peak oxygen consumption increased; ROS II in 60 minutes after stimulation, the duration of more than 150 minutes, the peak was significantly higher than that of peak oxygen consumption, without the phenomenon of.ROS peak 1 size and concentration of bacteria Is proportional to the peak time of no change; the ROS peak II with the increase of concentration of bacteria, the peak time was in advance, increased gradually. The peak lasted nearly 300 minutes in the reaction process, the luminescence signal with horseradish peroxidase were not detected in whole cells, that produced in the process of neutrophil cell membrane chemistry light permeability had no obvious change, the luminescence reaction mainly from biological and chemical reactions within the cell process. Obviously, the peak of ROS I and ROS II peak had distinct biological properties, representing two different functional states of the cell.
Experimental study of Pseudomonas aeruginosa in vitro also found that rat blood had obvious bacteriostasis, the role of neutrophils is directly related to, but has no direct relation with the blood plasma components. Antibacterial sterilization process can be divided into two distinct stages, namely the period of rapid bactericidal in ROS peak early and I the subsequent slow antibacterial sterilization period. In the period of rapid bactericidal bacteria inactivation rate is as high as 90%, this effect at the beginning of the reaction within 3 minutes after the basic completion. At this time, the degree of neutrophil phagocytosis of bacteria was not obvious, phagocytosis of neutrophil granulocyte proportion accounted for only 5%, obviously, the the extracellular mechanism of bactericidal and bacteriostatic phenomenon here with neutrophils. In the slow antibacterial sterilization period, neutrophil phagocytosis of bacteria with the prolonging of reaction time increased after the peak of ROS II (reaction After 200-250 minutes, the number of) the phagocytic rate of up to 30%-60%. phagocytosis of neutrophil ratio and unit granulocyte phagocytosis bacteria with the prolongation of the culture time and the degree of reaction system increased the number of viable bacteria in the reduction is similar, therefore, neutrophil phagocytosis and intracellular mechanism may play an important role in slow antibacterial sterilization period.
In summary, we believe that the peripheral blood neutrophils at least can be divided into two types, namely: easy foreign matter activation, phagocytosis and / or digestive activity with significant oxygen consumption early response of neutrophils and activated slowly, phagocytosis and / or digestive activity is not accompanied by oxygen consumption phenomenon of the slow response of neutrophils. In vivo anti infection process, extracellular mechanism is an important means of neutrophil killing. Neutrophil phagocytosis and intracellular mechanism mainly lies in the foreign bodies, such as the removal of pathogenic microorganisms in vivo. The mechanism of the reasons for the differences in the research on neutrophil killing mechanism of experimental results and now often think are discussed.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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