Siglec-E在脓毒症小鼠RAW264.7单核巨噬细胞的表达研究
发布时间:2018-05-05 04:11
本文选题:脓毒症 + Siglec-E ; 参考:《天津医科大学》2017年硕士论文
【摘要】:研究目的:唾液酸结合免疫球蛋白样凝集素(sialic-acid-binding-immunoglobulin- like lectins,siglec)最初被认定为细胞表面的跨膜受体,特异性表达于各种免疫细胞和造血细胞表面,能够抑制TLR信号,因此作为诊断和治疗各种疾病的高生物标记物引起了人们的关注。其家族的成员之一,siglec-E (与人同源的是siglec-7和siglec-9),主要表达于巨噬细胞和中性粒细胞表面,是急性炎症反应的重要负调节因子,是脓毒症治疗的潜在靶点。每年有800万患者因脓毒症而死亡,脓毒症仍是大部分住院患者最主要的死因,目前现有的治疗方法仍不是很有效。脓毒症时巨噬细胞的增殖、活化,功能状态对脓毒症的发展预后具有重要的作用。本研究通过LPS对小鼠RAW264.7单核巨噬细胞进行干预,观察脓毒症相关的siglec-E在巨噬细胞的表达及释放规律,摸索出较为合适的刺激浓度和作用时间,进一步探讨其与巨噬细胞分化和吞噬功能改变的关系,以及脓毒症时巨噬细胞吞噬功能障碍的机制,从而阐明LPS诱导巨噬细胞表达和释放siglec-E的规律以及其对巨噬细胞极化的影响,对脓毒症发生发展和预后的影响,寻找新的有效的治疗脓毒症的方法。研究方法:(1)常规培养小鼠RAW264.7单核/巨噬细胞株,选取对数期细胞,以2×106个/ml细胞密度接种,分别设置对照组和实验组。对照组不予处理,实验组予以LPS刺激,在不同的时间点(Oh、6h、12h、24h、36h、48h)分别提取对照组和实验组细胞的总RNA和总蛋白。通过实时荧光定量PCR法检测细胞裂解液中siglec-E mRNA表达量;通过Western blot法检测胞质中siglec-E蛋白含量。(2)常规培养小鼠RAW264.7单核/巨噬细胞株,选取对数期细胞,以2×106个/ml细胞密度接种,分别设置对照组和实验组。对照组不予处理,实验组细胞分别加入不同浓度的 LPS (O.O1ug/ml、0.05ug/ml、0.1ug/ml、1ug/ml、2ug/ml)刺激,孵育24h后,分别提取对照组和实验组细胞总RNA和总蛋白。通过实时荧光定量PCR法检测细胞裂解液中siglec-E mRNA表达;通过Western blot法检测胞质中siglec-E蛋白含量。结果:(1)在LPS刺激下,不同时间点的siglec-EmRNA表达量同对照组相比逐渐增加,6h~12h的表达量增加不明显,12h~36h的表达量明显增加,而36h~48h的表达量增加缓慢;siglec-E蛋白的表达量亦是如此。(2)不同剂量的LPS刺激下,siglec-E的mRNA表达量同对照组相比增高,在给予刺激浓度为O.01ug/ml~O.1ug/ml时的表达量明显增多,但增加速度缓慢;而在0.1ug/ml~1ug/ml时表达量明显增加,但在1ug/ml~2ug/ml浓度时表达量却有所下降;siglec-E的蛋白表达量与此大致一致。结论:(1)不同剂量的LPS刺激小鼠RAW264.7单核/巨噬细胞,可促进其表面siglec-E表达量增加,在0.01ug/ml~1ug/ml范围内表达量是增加的,但在1ug/ml的浓度时表达量显著,而在高于1ug/ml浓度下则产生了抑制,表达量下降。(2)LPS刺激后,随干预时间延长,小鼠单核巨噬细胞RAW264.7表面siglec-E表达量逐渐增加,在6h~48h之间表达量都是增加的,在48h时间点表达量最为显著。(3) siglec-E在脓毒症炎症时表达量增加且呈现一定的时间和剂量依赖。
[Abstract]:Research objectives: sialic-acid-binding-immunoglobulin- like lectins (siglec) is initially identified as a transmembrane receptor on the surface of the cell, specifically expressed on the surface of various immune cells and hematopoietic cells, and can inhibit the TLR signal as a high biomarker for the diagnosis and treatment of various diseases. One of the members of the family, siglec-E (homologous to siglec-7 and siglec-9), mainly expressed on the surface of macrophages and neutrophils, is an important negative regulator of acute inflammatory response and a potential target for the treatment of sepsis. 8 million patients die from sepsis and sepsis is still the majority of the year. The main causes of death in hospitalized patients are still not very effective at present. The proliferation, activation and functional status of macrophages in sepsis have an important role in the development of sepsis. This study was conducted through the intervention of RAW264.7 mononuclear macrophages in mice by LPS to observe the siglec-E of sepsis related to the macrophage In order to explore the relationship between macrophage differentiation and phagocytosis, and the mechanism of macrophage phagocytic dysfunction during sepsis, the regulation of LPS induced macrophage expression and release of siglec-E and its polarization to macrophages were elucidated. Influence on the development and prognosis of sepsis to find new effective methods for the treatment of sepsis. Study methods: (1) routine culture of mouse RAW264.7 mononuclear / macrophage strains, selected logarithmic phase cells, 2 x 106 /ml cells density inoculation, the control group and the experimental group were set respectively. The control group was not treated, the experimental group was stimulated by LPS, The total RNA and total protein of the cells in the control group and the experimental group were extracted at different time points (Oh, 6h, 12h, 24h, 36h, 48h). The expression of siglec-E mRNA expression in the cell lysate was detected by real time fluorescence quantitative PCR method, and the content of the protein in the cytoplasm was detected by Western blot. (2) the selected mononuclear / macrophage strains were selected as normal mice. The logarithmic phase cells were inoculated with 2 x 106 /ml cells, and the control group and the experimental group were set respectively. The control group was not treated. The cells of the experimental group were added with different concentrations of LPS (O.O1ug/ml, 0.05ug/ml, 0.1ug/ml, 1ug/ml, 2ug/ml) to stimulate 24h, and then the total RNA and total protein of the cells were extracted from the experimental group and the experimental group. The real-time fluorescence quantification was obtained by real-time fluorescence quantitative analysis. The expression of siglec-E mRNA in cell lysate was detected by PCR and the content of siglec-E protein in cytoplasm was detected by Western blot. Results: (1) the expression of siglec-EmRNA at different time points increased gradually under the stimulation of LPS, and the expression of 6h to 12h increased not obviously, and the expression amount of 12h to 36h was obviously increased. Increase slowly, the expression of siglec-E protein is also the same. (2) under the different doses of LPS stimulation, the expression of mRNA in siglec-E is higher than that of the control group, and the expression amount is significantly increased at the concentration of O.01ug/ml to O.1ug/ml, but the increase rate is slow, while the expression in 0.1ug/ml to 1ug/ml is significantly increased, but in 1ug/ml to 2ug/ml concentration. The expression of siglec-E was in accordance with this. Conclusion: (1) different doses of LPS stimulated the RAW264.7 mononuclear / macrophage in mice, which could increase the expression of siglec-E on the surface of the mouse, and increased in the range of 0.01ug/ml to 1ug/ml, but the expression was significant at the concentration of 1ug/ml, but higher than that of 1ug/ml concentration. (2) after LPS stimulation, the expression of siglec-E in the RAW264.7 surface of mononuclear macrophages increased gradually with the intervention time, and the expression level between 6h and 48h was increased. (3) the expression of siglec-E at the time of sepsis increased and showed a certain time. And dose dependent.
【学位授予单位】:天津医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R459.7
【参考文献】
相关期刊论文 前6条
1 薛明;徐静媛;刘玲;黄英姿;邱海波;;脓毒症相关免疫抑制:监测与挑战[J];中华重症医学电子杂志;2016年03期
2 刘旭;吕隽;李文强;;急诊脓毒症病死率评分在急诊脓毒症休克患者中的应用研究[J];医学临床研究;2016年02期
3 李楠;尤胜义;;脓毒症免疫功能紊乱研究进展[J];中国中西医结合外科杂志;2016年01期
4 王秦兰;王春梅;曹雪涛;;Siglec家族成员在免疫调控与免疫病理中作用的研究进展[J];中国肿瘤生物治疗杂志;2015年03期
5 肖为;杨明施;;脓毒症治疗的现状与新进展[J];医学综述;2014年08期
6 刘艳存;柴艳芬;姚咏明;;巨噬细胞在脓毒症发病机制中的作用研究进展[J];中国危重病急救医学;2013年04期
,本文编号:1846133
本文链接:https://www.wllwen.com/linchuangyixuelunwen/1846133.html
最近更新
教材专著