Notch信号在巨噬细胞调控重度烧伤创面中的作用

发布时间：2018-05-26 15:33

本文选题：烧伤 + RAW　；参考：《第四军医大学》2015年硕士论文

【摘要】：重度烧伤急救与治疗一直是临床和实验研究的重点和难点,伤后免疫功能的紊乱,不但可以促发全身性炎症反应综合征(Systemic inflammatory response syndrome,SIRS),而且还是多器官功能障碍(Multiple organ dysfunction syndrome,MODS)及患者死亡的主要诱因。巨噬细胞在整个烧伤炎症及损伤修复过程中均有重要的作用,是机体抗感染免疫的最主要效应细胞之一,同时也是机体内环境中免疫细胞和免疫分子致伤后免疫系统发生紊乱的重要细胞。目前关于调控巨噬细胞的免疫反应来控制伤后机体失控性炎症反应尚存不明确,因此从巨噬细胞免疫调节作用角度来救治重度烧伤是一个重要的解决思路。Notch分子首次发现于1917年的摩尔根实验室,因其突变能够造成果蝇翅膀边缘出现小缺口(Notches)而得名。目前已知,Notch分子信号家族成员具有高度的结构同源性及保守性,其受体及配体属于Ⅰ型膜蛋白,对哺乳动物而言Notch受体有4种(Notch 1、Notch 2、Notch3和Notch4),表达在多种组织和器官中。Notch配体分为Delta-like(Delta-like1,Delta-like3,Delta-like4)和Jagged(Jagged1,Jagged2)两类。Notch分子信号通路功能广泛,可以调控机体多种免疫细胞的发育及功能,而且可以通过多种分子机制调控单核-巨噬细胞系统的激活和功能。因此我们假设,notch分子信号通路在重度烧伤后存在表达上的变化并且对机体免疫反应应答发挥重要作用,本研究拟探讨其作用及分子机制。方法:1.课题实验研究第一部分建立稳定的c57/bl6j小鼠30%体表总面积(totalbodysurfacearea,tbsa)Ⅲ度烫伤模型,观察并评定创面深度,伤后依据不同时间点检测创面周围notch分子表达量;探讨重度烧伤情况下notch信号变化趋势。2.课题实验研究第二部分体外模拟重度烧伤后内环境,培养raw264.7巨噬细胞系,经不同烧伤血清刺激巨噬细胞,检测其notch信号表达改变;实验分假伤血清组和烧伤血清组,烧伤血清组可分为烧伤后6h、12h、24h、48h及4d、7d血清组。各组血清与rpmi-1640培养液制成20%的混合培养液,培养巨噬细胞,分别于0h、4h、8h、12h、24h、48h收集细胞样品,检测其notch1分子表达量。3.课题实验研究第三部分基本探讨重度烧伤后巨噬细胞notch信号通路激活的分子机制,并研究notch信号通路激活后对其分泌功能的影响。使用重度烧伤后24h及7d收集的血清刺激巨噬细胞,同时用假伤大鼠血清加脂多糖(lipopolysaccharide,lps)制成混合培养液刺激巨噬细胞,比较notch信号通路变化的异同;另外收集上述各组细胞培养上清液,检测巨噬细胞的分泌功能,elisa酶联反应试剂盒检测各组细胞培养上清液中il-6和tnf-α含量。结果:1.实验第一部分利用c57/bl6j小鼠建立稳定的30%tbsaⅢ度烫伤模型,伤后免疫组化染色结果显示:烧伤Ⅲ度12h及24h后notch1信号表达均明显高于对照组;而notch2分子表达量增高不明显;创周组织蛋白质免疫印迹结果显示:重度烧伤后创周组织中存在notch1和notch2分子的表达增高。2.实验第二部分体外模拟重度烧伤后内环境,培养利用raw264.7巨噬细胞系,经不同组重度烧伤血清刺激后,各组巨噬细胞中notch信号表达量有明显差异;其结果显示:利用早期重度烧伤血清制成混合培养液,培养巨噬细胞一定时间后,notch1分子表达量趋势总体升高,并且随着刺激时间延长而达到高峰;而采用重度烧伤后期的血清制成混合培养液,培养巨噬细胞相同时间后,对notch1信号的激活作用减弱。3.实验第三部分基本探讨重度烧伤后巨噬细胞notch信号通路激活的分子机制,并研究Notch 1信号通路激活后对其分泌功能的影响;研究结果表明重度烧伤巨噬细胞激活途径与LPS激活Notch信号机制相似,并且烧伤24 h血清、假伤血清+LPS刺激后培养上清液中炎症因子IL-6、TNF-α的含量明显增加,说明Notch信号激活后能够明显增强巨噬细胞的分泌功能。结论:Ⅲ度烫伤情况下创周存在Notch分子高表达;早期重度烧伤血清刺激下巨噬细胞Notch信号被激活,同时伴有IL-6及TNF-α分泌能力增强,且这现象的机制可能与LPS激活巨噬细胞Notch信号相似。
[Abstract]:First aid and treatment of severe burn have been the key and difficult point in clinical and experimental research. The disorder of immune function after injury can not only promote the Systemic inflammatory response syndrome (SIRS), but also the major organ dysfunction (Multiple organ dysfunction syndrome, MODS) and the main death of the patients. Macrophage plays an important role in the process of inflammation and repair of the whole burn. It is one of the most important cells in the body's anti infection immunity. It is also an important cell of the immune system after the injury of immune cells and immune molecules in the body. The inflammatory response to the body's runaway inflammation remains unclear, so the treatment of severe burns from the point of view of macrophage immunoregulation is an important solution for.Notch, which was first discovered in the Morgan laboratory in 1917 and named after its mutation can cause a small gap in the wings of Drosophila (Notches). Now, Notch The members of the molecular signal family have high structural homology and conservatism. Their receptors and ligands belong to type I membrane proteins. For mammals, there are 4 kinds of Notch receptors (Notch 1, Notch 2, Notch3 and Notch4), and the.Notch ligands in a variety of tissues and organs are divided into Delta-like (Delta-like1, Delta-like3, Delta-like4) and Jagged (Jagged1, and Jagged) Ged2) two kinds of.Notch signaling pathways are widely used to regulate the development and function of multiple immune cells, and can regulate the activation and function of monocyte macrophage system through a variety of molecular mechanisms. Therefore, we assume that the notch molecular signaling pathway changes in expression after severe burns and is immune to the body. This study should play an important role. This study intends to explore its role and molecular mechanism. Methods: 1. the first part of the study was to establish a stable totalbodysurfacearea (TBSA) model of c57/bl6j mice with total surface area (totalbodysurfacearea, TBSA) III degree scald, to observe and evaluate the depth of the wound, and to detect the expression of notch molecules around the wound after the injury. The study of the change trend of Notch signal in severe burn.2. second part of the experimental study on the internal environment of severe burn in vitro, the RAW264.7 macrophage system was cultured, the macrophage was stimulated by different burn serum to detect the change of the expression of Notch signal; the experimental group of false wound sera and burn serum, and the burn serum group could be divided into burn after burn. 6h, 12h, 24h, 48h and 4D, 7d serum group. The serum of each group and RPMI-1640 culture solution were made into 20% mixed culture medium, and the macrophages were cultured. The samples were collected in 0h, 4h, 8h, 12h, and 24h. The third part of the experimental study on the molecular expression of macrophage after severe burn was basically discussed. The effect of the activation of Notch signal pathway on its secretory function was studied. The serum stimulated by 24h and 7d after severe burn was used to stimulate macrophage, and the mixed culture solution of lipopolysaccharide (LPS) was used to stimulate macrophage, and the difference of Notch signaling pathway was compared. Cell culture supernatant was used to detect the secretory function of macrophages. The content of IL-6 and tnf- alpha in cell culture supernatant was detected by ELISA ELISA kit. Results: 1. the first part of the experiment used c57/bl6j mice to establish a stable 30%tbsa degree scald model. The results of immunohistochemical staining after injury showed that the Notch1 signal of 12h and 24h after the burn was 12h and 24h. The expression of Notch2 was significantly higher than that in the control group, but the expression of Notch2 molecules was not obvious. The results of the tissue protein immunoblotting showed that the expression of Notch1 and Notch2 molecules in the tissues of severe burns after severe burn.2. experiment was in vitro to simulate the internal environment of severe burn after burn, and the RAW264.7 macrophage system was used in different groups by different groups. After the burn serum was stimulated, the expression of Notch signal in the macrophages in each group was significantly different. The results showed that the mixed culture medium was made by the early severe burn serum, and the expression trend of Notch1 molecules increased after a certain time of macrophage culture, and reached the peak with the prolongation of the stimulation time; and the later period of severe burn was used. After a mixed culture of serum, the activation of Notch1 signal was weakened by the same time of macrophage, the third part of the activation of.3. signal was weakened, the molecular mechanism of activation of Notch signaling pathway in macrophages after severe burn was discussed, and the effect of Notch 1 signaling pathway on its secretory function was studied. The activation pathway of cell activation was similar to the mechanism of LPS activation of Notch signal, and 24 h serum was burned. The inflammatory factor IL-6 and TNF- alpha in the cultured supernatant were significantly increased after the stimulation of the false serum +LPS, indicating that the secretion function of the macrophages could be obviously enhanced after the activation of the Notch signal. Conclusion: the high expression of Notch molecules in the week of third degree scald; The Notch signal of macrophage was activated in early severe burn serum, and the secretion of IL-6 and TNF- alpha was enhanced, and the mechanism of this phenomenon may be similar to that of LPS activated macrophage Notch signal.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R644

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