本文選題：燒傷 + RAW��； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：重度燒傷急救與治療一直是臨床和實驗研究的重點和難點,傷后免疫功能的紊亂,不但可以促發(fā)全身性炎癥反應(yīng)綜合征(Systemic inflammatory response syndrome,SIRS),而且還是多器官功能障礙(Multiple organ dysfunction syndrome,MODS)及患者死亡的主要誘因。巨噬細(xì)胞在整個燒傷炎癥及損傷修復(fù)過程中均有重要的作用,是機(jī)體抗感染免疫的最主要效應(yīng)細(xì)胞之一,同時也是機(jī)體內(nèi)環(huán)境中免疫細(xì)胞和免疫分子致傷后免疫系統(tǒng)發(fā)生紊亂的重要細(xì)胞。目前關(guān)于調(diào)控巨噬細(xì)胞的免疫反應(yīng)來控制傷后機(jī)體失控性炎癥反應(yīng)尚存不明確,因此從巨噬細(xì)胞免疫調(diào)節(jié)作用角度來救治重度燒傷是一個重要的解決思路。Notch分子首次發(fā)現(xiàn)于1917年的摩爾根實驗室,因其突變能夠造成果蠅翅膀邊緣出現(xiàn)小缺口(Notches)而得名。目前已知,Notch分子信號家族成員具有高度的結(jié)構(gòu)同源性及保守性,其受體及配體屬于Ⅰ型膜蛋白,對哺乳動物而言Notch受體有4種(Notch 1、Notch 2、Notch3和Notch4),表達(dá)在多種組織和器官中。Notch配體分為Delta-like(Delta-like1,Delta-like3,Delta-like4)和Jagged(Jagged1,Jagged2)兩類。Notch分子信號通路功能廣泛,可以調(diào)控機(jī)體多種免疫細(xì)胞的發(fā)育及功能,而且可以通過多種分子機(jī)制調(diào)控單核-巨噬細(xì)胞系統(tǒng)的激活和功能。因此我們假設(shè),notch分子信號通路在重度燒傷后存在表達(dá)上的變化并且對機(jī)體免疫反應(yīng)應(yīng)答發(fā)揮重要作用,本研究擬探討其作用及分子機(jī)制。方法:1.課題實驗研究第一部分建立穩(wěn)定的c57/bl6j小鼠30%體表總面積(totalbodysurfacearea,tbsa)Ⅲ度燙傷模型,觀察并評定創(chuàng)面深度,傷后依據(jù)不同時間點檢測創(chuàng)面周圍notch分子表達(dá)量;探討重度燒傷情況下notch信號變化趨勢。2.課題實驗研究第二部分體外模擬重度燒傷后內(nèi)環(huán)境,培養(yǎng)raw264.7巨噬細(xì)胞系,經(jīng)不同燒傷血清刺激巨噬細(xì)胞,檢測其notch信號表達(dá)改變;實驗分假傷血清組和燒傷血清組,燒傷血清組可分為燒傷后6h、12h、24h、48h及4d、7d血清組。各組血清與rpmi-1640培養(yǎng)液制成20%的混合培養(yǎng)液,培養(yǎng)巨噬細(xì)胞,分別于0h、4h、8h、12h、24h、48h收集細(xì)胞樣品,檢測其notch1分子表達(dá)量。3.課題實驗研究第三部分基本探討重度燒傷后巨噬細(xì)胞notch信號通路激活的分子機(jī)制,并研究notch信號通路激活后對其分泌功能的影響。使用重度燒傷后24h及7d收集的血清刺激巨噬細(xì)胞,同時用假傷大鼠血清加脂多糖(lipopolysaccharide,lps)制成混合培養(yǎng)液刺激巨噬細(xì)胞,比較notch信號通路變化的異同;另外收集上述各組細(xì)胞培養(yǎng)上清液,檢測巨噬細(xì)胞的分泌功能,elisa酶聯(lián)反應(yīng)試劑盒檢測各組細(xì)胞培養(yǎng)上清液中il-6和tnf-α含量。結(jié)果:1.實驗第一部分利用c57/bl6j小鼠建立穩(wěn)定的30%tbsaⅢ度燙傷模型,傷后免疫組化染色結(jié)果顯示:燒傷Ⅲ度12h及24h后notch1信號表達(dá)均明顯高于對照組;而notch2分子表達(dá)量增高不明顯;創(chuàng)周組織蛋白質(zhì)免疫印跡結(jié)果顯示:重度燒傷后創(chuàng)周組織中存在notch1和notch2分子的表達(dá)增高。2.實驗第二部分體外模擬重度燒傷后內(nèi)環(huán)境,培養(yǎng)利用raw264.7巨噬細(xì)胞系,經(jīng)不同組重度燒傷血清刺激后,各組巨噬細(xì)胞中notch信號表達(dá)量有明顯差異;其結(jié)果顯示:利用早期重度燒傷血清制成混合培養(yǎng)液,培養(yǎng)巨噬細(xì)胞一定時間后,notch1分子表達(dá)量趨勢總體升高,并且隨著刺激時間延長而達(dá)到高峰;而采用重度燒傷后期的血清制成混合培養(yǎng)液,培養(yǎng)巨噬細(xì)胞相同時間后,對notch1信號的激活作用減弱。3.實驗第三部分基本探討重度燒傷后巨噬細(xì)胞notch信號通路激活的分子機(jī)制,并研究Notch 1信號通路激活后對其分泌功能的影響;研究結(jié)果表明重度燒傷巨噬細(xì)胞激活途徑與LPS激活Notch信號機(jī)制相似,并且燒傷24 h血清、假傷血清+LPS刺激后培養(yǎng)上清液中炎癥因子IL-6、TNF-α的含量明顯增加,說明Notch信號激活后能夠明顯增強(qiáng)巨噬細(xì)胞的分泌功能。結(jié)論:Ⅲ度燙傷情況下創(chuàng)周存在Notch分子高表達(dá);早期重度燒傷血清刺激下巨噬細(xì)胞Notch信號被激活,同時伴有IL-6及TNF-α分泌能力增強(qiáng),且這現(xiàn)象的機(jī)制可能與LPS激活巨噬細(xì)胞Notch信號相似。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R644

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