牛外周血中单核细胞和巨噬细胞及其TLRs家族基因的相关研究

发布时间：2018-06-28 00:20

本文选题：Toll样受体 + 牛　；参考：《扬州大学》2009年硕士论文

【摘要】： 单核细胞和巨噬细胞在抵抗和防御病原体感染中发挥了重要作用,尤其是在细胞内的病原体防御中发挥了巨大作用。根据组织内环境以及它们自身所处的不同分化阶段,它们都表现出不同的形态、机能和代谢作用。这些细胞可以通过细胞表面表达的TLR来识别和结合PAMP引发信号传导通路从而激发宿主的防御系统。至今为止,已经发现了TLR1-TLR10的部分基因序列,然而对牛外周血单核细胞及其巨噬细胞中Toll样受体的表达形式还不清除。因此本试验采取单核细胞以及由来巨噬细胞进行研究,主要研究牛外周血中此类细胞Toll样受体的表达形式,及其在LPS刺激条件下细胞因子的表达,从而得知该细胞在宿主免疫中发挥的重要作用。试验一牛外周血单核细胞和巨噬细胞分离培养和纯化方法本试验采用流式细胞分离仪和磁性细胞分离仪分离和鉴定了外周血中单核细胞及其由来的巨噬细胞,通过使用Repcell细胞培养板培养巨噬细胞,改进了Taflon Bag培养巨噬细胞的方法,且单核细胞和巨噬细胞的分离百分率分别为97.9%和73.9%。从而简化的了试验步骤,为以后的试验奠定了坚实的基础。试验二牛外周血单核细胞和巨噬细胞Toll样受体mRNA水平的表达形式通过RT-PCR和NIH光密度成像系统分析得到单核细胞和巨噬细胞TLRs mRNA的表达形式,并测得在LPS刺激时,TLRs家族中不同TLR对LPS的反应能力。研究发现,巨噬细胞在LPS刺激24h后,IL-6和TNF-αmRNA的表达量显著升高(P0.05),表达量分别为2.02±0.48和1.66±0.39。与此同时IL-8 mRNA的表达量相对提高,即2.14±0.52。TLR1、TLR3、TLR5、TLR8和TLR10的转录产物显著下降(P0.05),然而TLR2、TLR4、TLR6和TLR7则保持稳定。另外单核细胞的Toll样受体1-10以及细胞因子的表达量在LPS刺激前后保持稳定,其中TLR1、TLR2、TLR3、TLR4、TLR7、TLR8和TLR10的表达量高于1.0, IL-8和IL-6的表达量大于TNF-α,它们在LPS刺激后的表达量分别为1.38±0.41、1.47±0.17和1.21±0.51,与单核细胞相似的是TLR9不管是在正常培养条件下还是LPS刺激条件下都没有表达。试验结果表明,巨噬细胞在受到病原刺激后通过提高IL-6和TNF-αmRNA的表达量从而提高了天然免疫的作用。该研究为探讨巨噬细胞在天然免疫中的作用奠定了基础。第三章牛外周血单核细胞和巨噬细胞中Toll样受体蛋白质水平的表达形式本试验通过使用荧光免疫染色、流式细胞分离仪和共聚焦电镜,分析得知TLR2和TLR4在单核细胞和巨噬细胞表面的分布形式以及蛋白水平的表达量,并研究了在LPS刺激条件下,TLR2和TLR4的变化情况。试验结果表明,单核细胞和巨噬细胞在受到LPS刺激24h后,TLR2和TLR4蛋白水平表达量相对提高,分别为34.1%、47.1%和22.4%、10.1%,TLR2和TLR4蛋白水平的表达量与mRNA水平的表达形式一致,从而充分证实了TLR2和TLR4在单核细胞和巨噬细胞抵抗病原物质感染时发挥了重要作用。
[Abstract]:Monocytes and macrophages play an important role in resisting and defending pathogen infection, especially in intracellular pathogen defense. They all exhibit different morphological, functional and metabolic functions according to their internal environment and their own differentiation stages. These cells can recognize and bind PAMP triggered signal transduction pathway through TLR expressed on the cell surface to stimulate the host's defense system. Up to now, some gene sequences of TLR1-TLR10 have been found, but the expression of Toll-like receptor in bovine peripheral blood monocytes and macrophages has not been eliminated. Therefore, monocytes and macrophages were used to study the expression of Toll-like receptors and cytokines in bovine peripheral blood. Therefore, it is known that this cell plays an important role in host immunity. Isolation, culture and purification of bovine peripheral blood monocytes and macrophages the monocytes and their origin in peripheral blood were isolated and identified by flow cytometry and magnetic cell isolator. By using Repcell cell culture plate to culture macrophages, the method of Taflon bag culture was improved, and the isolation rates of monocytes and macrophages were 97.9% and 73.9%, respectively. Thus the test procedure is simplified and a solid foundation is laid for the later test. The expression of Toll-like receptor mRNA in bovine peripheral blood monocytes and macrophages was analyzed by RT-PCR and NIH optical density imaging system. The ability of different TLR to respond to LPS in the TLRs family was measured when stimulated by LPS. It was found that the expressions of IL-6 and TNF- 伪 mRNA in macrophages were significantly increased after 24 hours of LPS stimulation (P0.05), which were 2.02 卤0.48 and 1.66 卤0.39, respectively. At the same time, the expression of IL-8 mRNA increased relatively, that is, the transcription products of TLR3TLR5, TLR5, TLR8 and TLR10 decreased significantly (P0.05), but TLR2TLR4TLR6 and TLR7 remained stable. In addition, the expression of Toll-like receptor 1-10 and cytokines in monocytes remained stable before and after LPS stimulation. The expression levels of TLR1, TLR2, TLR4, TLR7TLR8 and TLR10 were higher than 1.0, IL-8 and IL-6 were higher than that of TNF- 伪, and the expression of TLR9 was 1.38 卤0.41 卤0.17 and 1.21 卤0.51respectively after LPS stimulation. TLR9 was not expressed in normal culture or LPS stimulated condition. The results showed that macrophages enhanced the innate immunity by increasing the expression of IL-6 and TNF- 伪 mRNA after pathogenetic stimulation. This study provides a basis for exploring the role of macrophages in innate immunity. The expression of Toll-like receptor protein in bovine peripheral blood monocytes and macrophages. The distribution of TLR2 and TLR4 on the surface of monocytes and macrophages and the expression of TLR2 and TLR4 on the surface of monocytes and macrophages were analyzed. The changes of TLR2 and TLR4 in LPS stimulated cells were studied. The results showed that the expression of TLR2 and TLR4 protein in monocytes and macrophages increased relatively after 24 hours of LPS stimulation, which were 34. 1% and 22. 4% respectively. The expressions of TLR2 and TLR4 in monocytes and macrophages were consistent with the expression of mRNA. Thus, TLR2 and TLR4 play an important role in monocyte and macrophage resistance to pathogen infection.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】