【作者】 高燕；

【导师】 刘佳佳；

【作者基本信息】 泸州医学院 ， 免疫学， 2009， 硕士

【摘要】 目的:中性粒细胞(polymorphonuclear neutrophil,PMN)是机体早期炎症反应中最重要的炎症细胞,它在炎症反应中处于核心地位。中性粒细胞来源于骨髓,属终末分化细胞。在正常和炎症部位的中性粒细胞,可组成性地经历自发性凋亡并被其它吞噬细胞识别、清除,在促进炎症的无损伤性收敛和维持机体内环境的稳定中发挥重要作用。细胞凋亡发生的原因和途径是复杂多样的,Bcl-2家族成员在细胞凋亡的基因调控过程中起着至关重要的作用,而Bax是Bcl-2家族中促凋亡的重要成员之一,它主要通过线粒体途径参与细胞凋亡的调控。Toll样受体(Toll-like receptor,TLRs)是一类识别病原相关分子模式(pathogen associated molecular pattern,PAMP)的模式识别受体,目前在人体内至少已发现TLR家族中的11个成员,分别被命名为TLR1～TLR11。TLRs广泛表达于各类组织细胞中,其中人类中性粒细胞是表达TLRs种类最多的细胞,表达除了TLR3以外所有的TLRs。尽管TLRs的配体都是PAMP,但不同的TLRs分子对PAMP的识别谱却不一样。虽然TLRs的PAMP识别对中性粒细胞生存的调控已有广泛研究,但通过不同TLRs识别PAMP对中性粒细胞凋亡的影响机制仍有待充分研究,特别是Bcl-2家族蛋白质在TLRs活化后对细胞的调控仍没有研究报道。因此,本研究采用中性粒细胞作为模型,对接受LPS、PGN、Loxoribine、Flaglline、Pam3CSK4、R-848和MALP-4七种不同TLR激动剂刺激的中性粒细胞中Bax蛋白的表达及其表达的差异性进行了研究,探讨TLR配体刺激下中性粒细胞内Bax蛋白表达水平,从而为确定Bcl-2家族蛋白质在TLR活化以后对中性粒细胞生存的影响提供进一步的实验依据。方法:1.抽取健康成人外周静脉血,Ficoll分离法分离、纯化中性粒细胞,台盼兰拒染试验和Wright-Giemsa混合染色后用光镜检测新鲜分离的中性粒细胞的活性和纯度;2.在分别加入LPS、PGN、Loxoribine、Flaglline、Pam3CSK4、R—848和MALP-4七种TLR激动剂和不加任何刺激物情况下培养,构建七种不同TLR激动剂作用后的细胞模型组和无刺激物作用的细胞对照组。3.提取不同培养模型组的中性粒细胞总蛋白,BCA法测定蛋白含量,SDS-PAGE观察各组蛋白表达差异性。4.使用特异性抗Bax多克隆抗体和内参GAPDH单克隆抗体通过Western bloting技术检测并比较Bax蛋白在不同TLR配体刺激下中性粒细胞模型中的表达水平。结果:1.光镜显示:新鲜分离、纯化的中性粒细胞活性和纯度均＞96%;2.培养12h后,将各组不同培养模型组的中性粒细胞裂解,抽提总蛋白进行SDS-PAGE并相互比较。结果显示,在不同刺激物刺激下,中性粒细胞蛋白质表达的种类和数量发生了不同程度的改变。3.TLR刺激物刺激中性粒细胞0h,2h,4h,8h,12h后,提取中性粒细胞蛋白,用Western blotting技术检测并比较Bax蛋白(24kDa)的表达水平。结果如下:LPS,PGN,loxoribine,flagellin,Pam3CSK43-1,1-dimethylureaR-848和MALP-2对中性粒细胞Bax蛋白有下调作用,且这七种TLR激动剂对Bax蛋白的表达差异无统计学意义。结论:1.LPS,PGN,loxoribine,flagellin,Pam3CSK4,R-848和MALP-2七种TLR激动剂能下调中性粒细胞内Bax蛋白的表达,这可能与Bax参与TLR激动剂诱导中性粒细胞死亡的信号传导途径有关。2.LPS,PGN,loxoribine,flagellin,Pam3CSK4,R-848和MALP-2七种TLR激动剂对Bax蛋白的表达差异无统计学意义,表明不同的TLR激动剂对Bax蛋白的表达影响与TLR受体激动剂类型可能无关。3.LPS,PGN,loxoribine,flagellin,Pam3CSK4,R-848和MALP-2七种TLR激动剂对延迟纯化中性粒细胞的自发性凋亡可能是有效的。更多还原

【Abstract】 Objective: Polymorphonuclear neutrophils (PMNs) are the most important effector cells in earlier inflammation and they play a core role in host infection against microbial infection. Neutrophils are terminally differentiated cells which come from marrow hemopoietic stem cell. Senescent neutrophils are recognized and phagocytized from nomal and inflammatory sites. Neutrophils undergo spontaneous apoptosis in vito and in vivo, and the progress has been recognized as a crucial mechanism for accomplishing resolution of inflammation and maintaining the stability of internal environment. During the study of apoptosis, an increasing number of reports have suggested that apoptosis is not exclusive form of programmed cell death (PCD). The cause and the pass of programmed cell death are very complicated. Bcl-2 family also plays a key role in regulating the cell apoptosis of neutrophils and Bax is one of the important regulated genes of apoptosis. Bax may regulate cell apoptosis by the pathway of the mitochondrion. TLRs, Toll-like receptors, are pattern recognition acceptors which recognize pathogen associated molecular pattern (PAMP). There are 11 members of TLRs, named TLR1-TLR11. Generally, TLRs are expressed in various kinds of rhagiocrine cell, and TLRs except TLR3 can be expressed in the human’s PMNs. TLRs ligand are PAMPs, but different TLRs recognize different PAMPs. A lot of studies about TLRs in PMNs are concentrated. But the research about TLRs in PMNs is at primary stage, specially, no study that the Bcl-2 family is activated by TLRs is reported. In order to explore new progress about neutrophil survival, the effects of TLR ligands on intracellular Bax (Bcl-2 homologous antagonist / killer) protein expression were studied. Various TLR ligands were used, such as LPS, PGN, Loxoribine, Flaglline, Pam3CSK4, R-848 and MALP-4. Methods: 1. Heparinized peripheral blood was obtained from healthy volunteers. Neutrophils were isolated using 3% dextran sedimentation followed by density gradient centrifugation with Ficoll. The purity of neutrophils population was > 96% with the Wright- Giemsa stain, and neutrophil viability was > 96%, as determined by trypan blue dye exclusion. 2. The neutrophils were cultured at the presence or absence of TLR ligands for the indicated time periods. The TLR ligands were LPS (TLR4), PGN (TLR2), loxoribine (TLR7), flagellin (TLR5), Pam3CSK4 (TLR1/2), R-848 (TLR7/8), MALP-2(TLR2/6). 3. The neutrophils were cultured in the presence or absence of TLR ligands for the indicated time periods. The whole cell lysates were subjected to SDS-PAGE. 4. Western blotting analysis was performed with specific Bax monoclonal antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody. To confirm equal amounts of loaded proteins, the membranes were reprobed with HRP-conjugated GAPDH monoclonal antibody as an internal control. Results: 1. Flow cytometry results suggested that the purity of neutrophil population was > 96%. 2. After 12 hours, the nature and quanlity of the PMNs cell protein was were changed. 3. Western blotting showed that the expression of Bax protein was down-regulated by TLR ligand that delayed apoptosis. The influence that different TLR ligands down-regulated the expression of Bax may have no relation with the type of TLR receptor. Conclusions: 1. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, can reduce the expression of Bax protein in neutrophils, which may be involved in TLR agonist-induced signal transduction pathway of neutrophil death. 2. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, were not statistically significant on the expression of Bax protein, indicating that the expression of Bax protein may be unrelated to different TLR agonists.3. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, may be effective at delying spontaneous apoptosis of PMN.更多还原