To Explore The Function Of Notch Signal Of Macrophages In The Severe Burn Wound

The severe burn is one of the most complicated clinical symptoms and the treatments of this kind of disease are intractable. It always accompanies the immune dysfunction or immunologic disorder: such as SIRS( Systemic inflammatory response syndrome) which is the leading cause of death. Macrophage plays a real important role during the whole burn process of inflammation and it is the major cell of anti-infection. However macrophage of internal environment also leads to immune system disorder which is on account of immune cells and immune molecules. To remedy this kind of uncontrolled inflammatory response by means of inflammatory regulation and control of the macrophage is still existing deficiency. Accordingly, we anticipate the treatments of severe burn through inflammation of the macrophages.The first discovery of Notch signal is in the laboratory of Morgan in 1917. The Notch molecular signal has highly structural homology and conservatism. For the mammal they have 4 receptors(Notch 1、Notch 2、 Notch 3 and Notch 4) and two kinds of ligands the one is Delta-like(Delta-like1, Delta-like3, Delta-like4) and the other kind is Jagged(Jagged 1,Jagged 2). According to modern scientific research, the Notch molecular signaling pathway has widely function. So we assume to research the role of Notch signal in severe burns, and discuss the molecular mechanism.To investigate the dynamic expression of Notch 1 signal and the secretion of interleukin 6(IL-6) and tumor necrosis factor α(TNF-α) from macrophage RAW264.7 cells induced by burn serum, we designed the following experiment. Methods:1. The part one of the experimentation: establish a stable scalded model of C57/BL6 J of 30%TBSA( Total body surface area). Observe and assess the tissues around burn wound and detect the Notch signal expression in the wound.2. The part two of the experimentation: Twenty-four male Sprague Dawley rats were divided into the sham burn group and burn groups randomly, each group had eight rats. The mice of burn groups were made the full-thickness thermal burns comprising 30% total body surface area. The serum of burn rats were collected after burn 6 h、12 h、24 h、48 h、4 d and 7 d. The sham burn groups employed 37℃ water-bath and treated the serum as control. The mixed culture medium contained 20% burned serum, RAW264.7 cells were cultivated with each groups of burned serum for 0, 4, 8, 12, 24, 48 h and then detect the Notch signal expression of the macrophage.3. The part three of the experimentation: use the serum of burn rats which are after injury 24 h and 7 d, and the sham burn serum. The mixed culture medium contained 20% burned serum, RAW264.7 cells were cultivated with each groups of burned serum for 0, 4, 8, 12, 24, 48 h. In addition, 100 ng/m L LPS+20% sham burn serum were applied to culture medium and employed it to cultivate RAW264.7. The Notch 1 signaling of macrophages were detected after cultivation and the secretion of IL-6 and TNF-α in culture supernatants of each group was measured by ELISA. Results:1. In the full-thickness thermal burns skins, the result of immunofluorescent stainningshows that the expression of Notch 1 signal is higher than the control group, however the expression of Notch 2 is unconspicuous. And the result of western blot shows that both the expression level of Notch1 and Notch2 are higher than the control group around the burn skin areas.2. The level of Notch 1 signal had no obvious difference at 0, 4, 8, 12, 24, 48 h after stimulation of sham burn serum. In burn at 6 h、12 h、24 h、48 h group, the level of Notch 1 signaling was obviously high and as time progresses the signal peaked. In burn at 4 d and 7 d group, the Notch 1 signal were flat as the sham burn serum group.3. In the mixed culture medium of LPS+sham burn serum group, after stimulation the level of Notch 1 signaling was as high as the burn at 24 h groups. The contents of IL-6 and TNF-α in burn at 24 h group and in LPS+sham burn serum group were significantly higher than in the sham burn serum group and the burn at 7 d group. Conclusion:The Notch signaling and the secretion of IL-6 and TNF-α of macrophage could be stimulated by burn serum and this kind of high level signaling could be imitated by LPS.