内质网应激通过调控巨噬细胞向M1方向极化产生致炎作用的实验研究

发布时间：2018-11-13 12:12

【摘要】：目的:脓毒症(sepsis)是常见的致死原因,其病理过程常伴随严重的炎症反应,已有学者发现脓毒症最常见的致病因素——脂多糖(lipopolysaccharide,LPS)可通过诱导巨噬细胞向M1型极化进而产生炎症反应。内质网应激(Endoplasmic reticulum stress,ERS)是机体遭受到外界刺激(内毒素,缺氧等)时内质网产生的一个复杂的适应性反应,可通过调控多条炎症反应通路产生致炎作用。由于内质网应激和巨噬细胞极化均可控制炎症反应并且二者也有相似的诱发因素,因此二者是否存在关联是本实验探究的主要内容。方法:第一部分实验用革兰阴性菌内毒素脂多糖腹腔注射造成炎症反应,6h后取小鼠肺组织,用western blot检测内质网应激相关蛋白eIF2α,p-eIF2α,IRE1α,p-IRE1α,p-IκB,XBP-1的表达量来判断脓毒症所致的炎症反应是否与内质网应激有关。第二部分实验将小鼠分为四组,取出其中两组分别注射内质网应激激动剂TM和内质网应激抑制剂TUDCA预处理30min,然后将处理后的两组和未处理的一组分别注射等量的LPS,最后一组注射PBS作为对照。通过流式细胞仪检测巨噬细胞M1的标记物iNOS的表达情况来判断巨噬细胞极化是否与内质网应激有关。第三部分实验取小鼠骨髓间充质干细胞用M-CSF定向诱导至巨噬细胞M0,然后用LPS刺激使其发生极化,然后用RT-PCR检测巨噬细胞M1因子IL-1,IL-12,iNOS,TNF-α的表达水平来检测极化的细胞是否为M1型。第四部分实验将定向诱导的巨噬细胞M0分为6组:M-CSF组;LPS组;LPS+TUDCA(50μg/m L)组;LPS+TUDCA(100μg/m L)组;LPS+TUDCA(200μg/mL)组,LPS+TG组。内质网应激激动剂TG和抑制剂TUDCA均需预处理30min。在体外条件下通过流式细胞仪检测巨噬细胞M1的标记物iNOS的表达情况来判断巨噬细胞极化是否与内质网应激有关并且观察巨噬细胞极化程度是否与内质网应激抑制程度有关。结果:第一部分实验相较于对照组,实验组内质网应激相关蛋白e IF2α,p-e IF2α,p-IRE1α,p-IκB,XBP-1的表达量均升高。第二部分实验巨噬细胞M1的标记物iNOS在LPS组表达高于对照组(PBS组);相较于LPS组内质网应激激动剂TM预处理组iNOS表达量增加,而内质网应激抑制剂TUDCA预处理组,iNOS表达量降低。第三部分体外实验用LPS诱导极化的巨噬细胞高表达IL-1,IL-12,iNOS,TNF-α四种巨噬细胞M1的标记物。第四部分实验巨噬细胞M1的标记物iNOS的表达量在LPS组,LPS+TUDCA(50μg/m L)组,LPS+TUDCA(100μg/m L)组,LPS+TUDCA(200μg/m L)依次递减,用内质网应激激动剂TG预处理组,iNOS表达量最高。结论:通过体内体外实验证实在LPS所致的脓毒症模型中,内质网应激通过调节巨噬细胞向M1型极化产生致炎作用。
[Abstract]:Objective: sepsis (sepsis) is a common cause of death, and its pathological process is often accompanied by severe inflammatory reaction. Some scholars have found that the most common pathogenic factor of sepsis is lipopolysaccharide (lipopolysaccharide,). LPS) can induce macrophage to M 1 polarization and then produce inflammatory response. Endoplasmic reticulum stress (Endoplasmic reticulum stress,ERS) is a complex adaptive response of endoplasmic reticulum (ER) induced by external stimuli (endotoxin, hypoxia, etc.), which can induce inflammation by regulating multiple inflammatory response pathways. Because endoplasmic reticulum stress and macrophage polarization can both control inflammatory response and have similar inducing factors, the relationship between them is the main content of this experiment. Methods: in the first part of the experiment, inflammatory reaction was induced by intraperitoneal injection of lipopolysaccharide (LPS) from Gram-negative bacteria. Lung tissues were taken from mice 6 hours later. Endoplasmic reticulum stress-related proteins eIF2 伪, p-eIF2 伪, IRE1 伪, p-I 魏 B were detected by western blot. The expression of XBP-1 was used to determine whether the inflammatory response induced by sepsis was related to endoplasmic reticulum stress. In the second part of the experiment, mice were divided into four groups. Two groups were pretreated with endoplasmic reticulum stress agonist (TM) and endoplasmic reticulum stress inhibitor (TUDCA) for 30 min, respectively. Then the treated group and the untreated group were injected with the same amount of LPS, respectively. The last group was given PBS as a control group. The expression of iNOS, a marker of macrophage M1, was detected by flow cytometry to determine whether the polarization of macrophage was related to endoplasmic reticulum stress. In the third part of the experiment, murine bone marrow mesenchymal stem cells were induced to macrophage M0 by M-CSF, then polarized by LPS stimulation, and then detected by RT-PCR for IL-1,IL-12,iNOS, of macrophage M1 factor. The expression level of TNF- 伪 was used to detect whether the polarized cells were M 1 type. In the fourth part of the experiment, we divided the directed macrophage M0 into six groups: M-CSF group, LPS group; LPS TUDCA (50 渭 g / mL group,; LPS TUDCA (100 渭 g / mL group (; LPS TUDCA (200 渭 g/mL group) group. Endoplasmic reticulum stress agonist (TG) and inhibitor TUDCA were pretreated for 30 min. The expression of iNOS, a marker of macrophage M1, was detected by flow cytometry in vitro to determine whether the polarization of macrophage was related to endoplasmic reticulum stress and whether the degree of macrophage polarization was related to the inhibition of endoplasmic reticulum stress. Results: compared with the control group, the expression of ER stress-related proteins e IF2 伪, p-IRE1 伪, p-I 魏 B-1 XBP-1 increased in the experimental group. In the second part, the expression of macrophage M1 marker iNOS in LPS group was higher than that in control group (PBS group). Compared with LPS group, the expression of iNOS was increased in TM preconditioning group, but decreased in TUDCA pretreated with endoplasmic reticulum stress agonist (TUDCA) group. In the third part, LPS was used to induce the overexpression of IL-1,IL-12,iNOS,TNF- 伪 four kinds of macrophages M1 markers. In the fourth part, the expression of iNOS, the marker of macrophage M1, was decreased in the LPS group (, LPS TUDCA (50 渭 g / mL) group (, LPS TUDCA (100 渭 g / mL) group (, LPS TUDCA (200 渭 g / mL) group, and was pretreated with the endoplasmic reticulum stress agonist TG. The expression of iNOS was the highest. Conclusion: in vivo and in vitro experiments demonstrated that endoplasmic reticulum stress induced inflammation by regulating macrophage polarization to M1 type in LPS induced sepsis model.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

【相似文献】