DOCK2对中性粒细胞凋亡和表达TLR4的影响

发布时间：2018-09-09 18:58

【摘要】： 目的：中性粒细胞(polymorphonuclear neutrophils, PMNs)是机体最重要的炎症细胞,它在机体固有性免疫应答中具有重要作用,其胞浆内富含的细胞毒性物质既可杀伤外来入侵的病原微生物,也可造成自身组织细胞的损伤。研究表明,中性粒细胞粘附、活化及凋亡的整个过程可受Toll样受体(Toll-like receptors, TLRs)的调节,促成炎症反应的无损伤性收敛,使机体维持内环境的稳态。其中,TLR4是人们最早发现的能够直接介导机体与病原体反应的Toll样受体。最近,日本科学家发现一种与中性粒细胞的趋化有关的蛋白DOCK2 (Dedicator of cytokinesis 2),其聚集到中性粒细胞内朝着感染源的一侧,使中性粒细胞改变自身形态,更高效地向感染源移动。本课题主要探讨DOCK2与中性粒细胞凋亡的相关性以及DOCK2与中性粒细胞上表达的TLR4之间的联系。这为今后更深入研究中性粒细胞抗感染能力的机制提供了潜在的依据,也为临床上治疗炎症性相关疾病提供了新的靶点。方法：抽取健康成人外周静脉血,Ficoll密度梯度法分离、纯化中性粒细胞,并利用台盼蓝拒染试验、怀特-姬姆萨混合染色法鉴定细胞活力与纯度均达96%以上,按实验需求构建不同的实验模型：实验组(DOCK-2刺激组)、阳性对照组(IL-1β刺激组、TNF-α刺激组)、阴性对照组(空白组)。运用光学显微镜观察不同培养状态下的细胞形态；用流式细胞术检测不同组中性粒细胞的凋亡率；通过反转录-聚合酶链反应(RT-PCR)技术检测TLR4mRNA在各组中的表达情况；免疫印迹(western blotting)技术检测各组中TLR4的表达情况。结果：1.运用流式细胞术检测DOCK2对PMNs凋亡是否有影响,结果显示：与空白组比较,DOCK2组PMN凋亡率是下降的,具有明显的差异性(P0.05)。2.RT-PCR技术检测PMNs在不同药物刺激组中TLR4 mRNA的表达情况。结果显示：同空白组相比,DOCK2组TLR4 mRNA的表达上调,且有明显的差异性(P0.01)。3.培养6 h后,将各组模型组中的中性粒细胞裂解,提取总蛋白进行SDS-PAGE并相互比较。结果显示：在不同刺激物刺激下,中性粒细胞上TLR4蛋白质表达的性质和数量发生了不同程度的改变。4.不同实验组刺激PMN 6 h后,提取蛋白并用Western blotting检测TLR4蛋白的表达。结果显示：同空白组相比,DOCK2组TLR4蛋白的表达是上调的,有明显的差异性(P0.01)。结论：1.DOCK2可能对PMNs的凋亡有延迟作用。2.DOCK2可能对中性粒细胞上TLR4的表达有上调作用。3.TLR4可能抑制PMN的凋亡。4.DOCK2抑制PMNs凋亡的同时上调TLR4的表达。但也可能前者引起后者,或者后者引起前者。并且,二者变化的量之间可能存在一定的关系。
[Abstract]:Objective: neutrophil (polymorphonuclear neutrophils, PMNs) is the most important inflammatory cell in the body. It plays an important role in the innate immune response of the body. The cytotoxic substances abundant in the cytoplasm of neutrophil (polymorphonuclear neutrophils, PMNs) can kill the invading pathogenic microorganisms. They can also cause damage to their own tissues and cells. The study shows that the whole process of neutrophil adhesion activation and apoptosis can be regulated by Toll like receptor (Toll-like receptors, TLRs) which can promote the noninvasive convergence of inflammatory response and make the body maintain the homeostasis of the internal environment. TLR4 is the first Toll-like receptor that can directly mediate the reaction between organism and pathogen. Recently, Japanese scientists have discovered a protein DOCK2 (Dedicator of cytokinesis 2 associated with neutrophil chemotaxis, which gathers to the side of the neutrophil towards the source of infection, causing the neutrophil to change its morphology and move more efficiently to the source of infection. The purpose of this study was to investigate the relationship between DOCK2 and neutrophil apoptosis and the relationship between DOCK2 and TLR4 expressed on neutrophils. This provides a potential basis for the further study of the mechanism of neutrophil anti-infection, and provides a new target for clinical treatment of inflammatory diseases. Methods: neutrophils were isolated and purified by Ficoll density gradient method from peripheral venous blood of healthy adults. The viability and purity of neutrophils were identified by trypan blue exclusion assay and White-Giemsa mixed staining method. Different experimental models were constructed according to experimental demands: experimental group (DOCK-2 stimulation group), positive control group (IL-1 尾 stimulation group), negative control group (blank group). The apoptosis rate of neutrophils in different groups was detected by flow cytometry and the expression of TLR4mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot (western blotting) technique was used to detect the expression of TLR4 in each group. The result is 1: 1. The effect of DOCK2 on PMNs apoptosis was detected by flow cytometry. The results showed that the apoptosis rate of PMN in DOCK2 group was lower than that in control group (P0.05). 2. RT-PCR was used to detect the expression of TLR4 mRNA in different drug stimulated groups. The results showed that the expression of TLR4 mRNA was up-regulated, and there was significant difference (P0.01). 3. After 6 hours of culture, the neutrophils in each group were lysed and the total protein was extracted and compared with each other. The results showed that the nature and quantity of TLR4 protein expression on neutrophils changed in varying degrees under different stimuli. After 6 h stimulation of PMN in different experimental groups, the expression of TLR4 protein was detected by Western blotting. The results showed that the expression of TLR4 protein was up-regulated in the control group (P0.01). Conclusion: 1. DOCK2 may delay the apoptosis of PMNs. 2. DOCK2 may up-regulate the expression of TLR4 in neutrophils. 3. TLR4 may inhibit the apoptosis of PMN. 4. DOCK2 inhibits the apoptosis of PMNs and up-regulates the expression of TLR4. But the former may cause the latter, or the latter may cause the former. Moreover, there may be a certain relationship between the quantity of the two changes.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.12

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