NaCl对M1型巨噬细胞极化的调控及其机制研究

发布时间：2018-06-14 00:54

  本文选题：NaCl + LPS　； 参考：《济南大学》2017年硕士论文

【摘要】：目的:研究高盐对M1型巨噬细胞极化的调控,并在内毒素休克炎症疾病模型中得到验证,这为临床一些疾病的干预和治疗提供了理论依据,同时为我们下一步探究其机制打好坚实的基础。方法:1.用GM-CSF诱导小鼠骨髓来源的原代巨噬细胞(BMDMs),在加入LPS(200 ng/ml)和IFN-γ(10 ng/ml)之前,用不同浓度的NaCl预处理BMDMs 2小时。(1)用CCK8检测细胞活力的方法检测不同浓度的NaCl对M1型巨噬细胞的活力的影响,确定一个合适的高盐浓度用于后续的实验。(2)用该合适的高盐浓度预处理BMDMs,分别在处理后的6、12和24 h收取细胞培养上清,用ELISA检测M1型巨噬细胞因子IL-6和IL-12p40的表达。(3)用不同浓度的Na Cl(5、10和20 mM)预处理BMDMs,24小时后,收取细胞并提取总RNA,RT-PCR检测IL-6和IL-12p40在mRNA水平的表达。(4)流式细胞术检测胞内抗体IL-12p40的平均荧光强度。(5)流式细胞术检测M1型巨噬细胞表面标记CD86和MHCII的表达。(6)Western Blot检测IRF5蛋白的表达。2.成功诱导BMDMs,将细胞分为两组,其中一组用NaCl预处理6小时,另外一组细胞不做任何处理,收取细胞,使其浓度为1×107/ml。将20只老鼠分为两组,每组10只,分别将200μl上述两组细胞腹腔注射入小鼠体内。(1)24小时后,每只小鼠腹腔注射800μg的LPS,观察小鼠死亡率。(2)24小时后,每只小鼠腹腔注射200μg的LPS,12小时后小鼠眼球取血,断颈处死小鼠,抽取腹腔细胞,获得小鼠肺、肝脏和脾。(1)病理切片HE染色显示小鼠肺和肝脏的损伤情况。(2)ELISA检测小鼠血清中IL-6和IL-12p40的表达。(3)RT-PCR检测小鼠腹腔和脾细胞中IL-6和IL-12p40在mRNA水平的表达。结果:1.(1)高浓度的NaCl(≥40 mM)减弱了M1型巨噬细胞的活力。合适的高盐浓度为20 mM,该浓度不会随着时间的增加影响细胞活力。(2)ELISA结果显示,NaCl预处理后,IL-6和IL-12p40的表达显著降低,并且具有时间和剂量的依赖性。(3)RT-PCR显示,与对照组相比,NaCl预处理组IL-6和IL-12p40的表达明显降低了,且具有剂量依赖性。(4)流式细胞术显示,相对于正常组,NaCl预处理组IL-12p40的表达也显著减少。(5)流式细胞术显示,与正常组相比,NaCl预处理组M1型巨噬细胞表面标记CD86和MHCII的表达并没有明显的变化。(6)Western Blot显示,与对照组相比,NaCl预处理组IRF5的表达也显著减少。2.(1)注射NaCl预处理的BMDMs组小鼠的死亡率明显降低。(2)HE染色显示,NaCl干预可明显抑制LPS所致的肺和肝脏组织的损伤。(3)注射NaCl预处理的BMDMs小鼠体内,LPS所致的M1型巨噬细胞的极化明显减少,其M1型巨噬细胞因子IL-6和IL-12p40的表达显著减少。结论:1.NaCl可抑制体外M1型巨噬细胞的极化,其细胞因子IL-6和IL-12p40的表达显著降低,其机制可能是NaCl减少了M1型巨噬细胞的转录因子IRF5的表达。2.在小鼠体内,NaCl减少了M1型巨噬细胞的极化,从而减轻了LPS所致的内毒素损伤,减弱了LPS所引起的炎症反应。
[Abstract]:Objective: to study the effect of high salt on the polarization of M1 macrophages and to verify it in the model of inflammatory diseases of endotoxic shock, which provides a theoretical basis for the intervention and treatment of some diseases. At the same time for our next step to explore its mechanism to lay a solid foundation. Method 1: 1. Mouse bone marrow-derived primary macrophages were induced by GM-CSF. Before the addition of LPS200 ng / ml and IFN- 纬 10 ng / ml, BMDMs were pretreated with different concentrations of NaCl for 2 hours. 1) the effects of different concentrations of NaCl on the activity of M1 macrophages were detected by CCK8 method. To determine a suitable concentration of high salt for further experiment. (2) to pretreat BMDMswith the appropriate concentration of high salt, and to collect the supernatant of cell culture at 6h and 24h after treatment, respectively. The expressions of IL-6 and IL-12p40 in M1 macrophage were detected by Elisa. The expression of IL-6 and IL-12p40 at mRNA level was detected by RT-PCR. Flow cytometry was used to detect the average fluorescence intensity of intracellular antibody IL-12p40. Flow cytometry was used to detect the expression of CD86 and MHCII on the surface of M1 macrophages. Western blot was used to detect the expression of IRF5 protein. BMDMswere successfully induced and divided into two groups. One group was pretreated with NaCl for 6 hours, the other group received cells without any treatment, and the concentration of the cells was 1 脳 10 ~ (7) 路ml ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). Twenty mice were divided into two groups, 10 rats in each group. After intraperitoneal injection of 200 渭 l cells into mice for 24 hours, each mouse was injected intraperitoneally with 800 渭 g LPSs. The mortality of mice was observed after 24 hours. Each mouse was injected intraperitoneally with 200 渭 g LPS for 12 hours. The mice were killed by amputation of their necks, the peritoneal cells were extracted, and the lungs of the mice were obtained. The expression of IL-6 and IL-12p40 in serum was detected by Elisa. RT-PCR was used to detect the mRNA expression of IL-6 and IL-12p40 in peritoneal and splenic cells of mice. Results the activity of M1 macrophages was weakened by high concentration of NaCl1 (鈮，

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