本文選題：異常黑膽質(zhì)成熟劑 + 燒傷�。� 參考：《新疆醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的：通過構(gòu)建大鼠梳狀燙傷模型模擬二度以上燒傷創(chuàng)面早期進(jìn)行性加深的病理生理過程,系統(tǒng)觀察不同劑量異常黑膽質(zhì)成熟劑(Abnormal Savda Munziq,ASMq)對燒傷創(chuàng)面進(jìn)行性加深過程的作用,探討潛在的劑量效應(yīng)及相關(guān)作用機(jī)制。方法：首先構(gòu)建大鼠燒傷創(chuàng)面進(jìn)展模型,即以200～220g Sprague-Dawley (SD)大鼠為研究對象,戊巴比妥鈉麻醉后,將訂制的長方體銅塊(切面為20mm×10mm)浸于100℃水中5分鐘,再置于脫毛后的大鼠背部皮膚持續(xù)20秒,間隔5mm建立四個(gè)創(chuàng)面,取創(chuàng)面間隙及部分創(chuàng)面組織(20mm×9mm)為淤滯區(qū)代表進(jìn)行研究。在通過不同時(shí)間段大體及HE染色觀察確認(rèn)燒傷創(chuàng)面早期進(jìn)展性變化。再利用RT-PCR、ELISA、TUNEL染色、免疫組化/熒光染色等方式,從分子水平探討燒傷創(chuàng)面早期進(jìn)展性加深的機(jī)制,并找出可能的干預(yù)時(shí)間點(diǎn)。其次,利用不同劑量ASMq灌胃給藥,從組織學(xué)、分子水平觀察其對早期燒傷創(chuàng)面的作用,及其對相關(guān)損傷機(jī)制的影響。最后利用特異性分子信號(hào)抑制劑來明確可能介導(dǎo)ASMq保護(hù)作用的信號(hào)通路。結(jié)果：第一部分實(shí)驗(yàn)中,根據(jù)大體觀及HE染色顯微鏡下觀察,我們發(fā)現(xiàn)深二度燒傷后,大鼠淤滯區(qū)充血并呈壞死傾向,可見兩直接燒傷損傷創(chuàng)面呈融合趨勢。HE染色后鏡下觀察顯示,燒傷后72小時(shí)內(nèi),隨著時(shí)間變化,大鼠燒傷創(chuàng)面淤滯區(qū)皮膚組織內(nèi)氧化應(yīng)激水平逐步升高,內(nèi)源性抗氧化酶系(GPx,SOD)水平因消耗增加而較正常對照組明顯降低(P0.05)。燒傷后48小時(shí),氧化應(yīng)激水平達(dá)到高峰。與此同時(shí),自由基產(chǎn)生的主要兩條途徑的關(guān)鍵酶--黃嘌呤氧化酶(Xanthine Oxidase, XO)及屬于還原型煙酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶的非吞噬細(xì)胞氧化酶4(non-phagocytic cell oxidase 4, Nox4)在燒傷后均呈現(xiàn)明顯的表達(dá)增加(P0.05)。燒傷后大鼠淤滯區(qū)組織中炎癥介質(zhì)(MPO, IL-1β, IL-6)的分布與釋放隨時(shí)間明顯增加(P0.05),48小時(shí)時(shí)最為明顯。而淤滯區(qū)組織中活化的NF-κB表達(dá)也隨時(shí)間表達(dá)增加(P0.05),各時(shí)間組與正常對照組均有顯著差異。此外,燒傷后淤滯區(qū)皮膚組織出面明顯的細(xì)胞凋亡增加,伴隨著活化的凋亡相關(guān)蛋白-裂解的含半胱氨酸的天冬氨酸蛋白水解酶3/9(cleaved cysteinyl aspartate specific proteinase, Cleaved Caspase 3/9,CC3/9)表達(dá)上調(diào)。第二部分實(shí)驗(yàn)中,我們觀察到ASMq治療可以有效減輕淤滯區(qū)皮膚組織損傷。此外,ASMq劑量相關(guān)的降低組織內(nèi)MDA水平,并顯著提高燒傷后降低的內(nèi)源性抗氧化酶系(GPx, SOD)水平(P0.05),同時(shí)劑量相關(guān)的降低XO及Nox4表達(dá)(P0.05)。ASMq還可緩解淤滯區(qū)組織中炎癥介質(zhì)釋放(P0.05),并上調(diào)NF-κB活化水平(P0.05),高劑量的ASMq治療作用更加明顯,而LPS干預(yù)可以逆轉(zhuǎn)高劑量ASMq的治療作用。另外,ASMq治療后淤滯區(qū)組織細(xì)胞凋亡明顯減少,CC3/9的分布與表達(dá)也明顯降低(P0.05),ASMq作用呈劑量依賴效應(yīng)；Erk激活阻滯劑PD98059有效逆轉(zhuǎn)高劑量ASMq治療對淤滯區(qū)組織細(xì)胞凋亡的緩解作用及CC3/9表達(dá)下調(diào)(P0.05)。第三部分,我們調(diào)查了對可能參與ASMq作用的信號(hào)通路,實(shí)驗(yàn)結(jié)果結(jié)果顯示：1)ASMq治療可以減少NF-κB活化,中、高劑量ASMq作用更加顯著,高劑量最為明顯(P0.01)；2)ASMq治療可以影響線粒體相關(guān)凋亡信號(hào)通路中Bad磷酸化及Bcl-xL.細(xì)胞色素C(Cytochomre C, Cyto C)的表達(dá)。高、中、低劑量ASMq治療均可以明顯增加Bad磷酸化(P0.01),呈劑量相關(guān)作用(P0.01)。而對于Bcl-xL,中、高劑量的ASMq的上調(diào)作用更加明顯。中、高劑量ASMq治療可以顯著下調(diào)燒傷引起的Cyto C表達(dá)增加,高劑量的作用更加明顯。高劑量ASMq對Bad磷酸化及Bcl-xL、細(xì)胞色素C(Cyto C)的表達(dá)的影響,均可依被PD98059逆轉(zhuǎn),提示Erk激活在ASMq作用中起重要調(diào)節(jié)作用；3)根據(jù)已有的文獻(xiàn),ASMq可能對Ras/Erk/p90RSK信號(hào)級聯(lián)存在調(diào)節(jié)作用,而這一級聯(lián)也是線粒體凋亡信號(hào)通路上游重要的調(diào)節(jié)信號(hào)級聯(lián)。免疫熒光染色顯示,隨著劑量增加,ASMq治療可以顯著提高磷酸化的Erk (p-Erk)分布,而Western blotting結(jié)果也顯示,低、中、高劑量ASMq均可進(jìn)一步顯著增加Erk磷酸化激活,并呈劑量依賴增加(P0.01),PD98059可以顯著抑制ASMq對Erk的激活作用(P0.01)。此外,中、高劑量ASMq均可顯著上調(diào)Ras表達(dá)水平,且高劑量組作用更加明顯(P0.01),而PD98059對Ras表達(dá)沒有影響(P0.05)。高、中、低劑量的ASMq對p90RSK的表達(dá)均有顯著的上調(diào)作用,與Erk磷酸化激活相對應(yīng),并呈劑量相關(guān)作用,而Erk抑制劑PD98059可顯著下調(diào)高劑量ASMq增加的p90RSK表達(dá)水平。結(jié)論：(1)早期的二度以上燒傷創(chuàng)面存在一個(gè)動(dòng)態(tài)的進(jìn)行性加深過程,初始創(chuàng)面附近皮膚組織有損傷進(jìn)展擴(kuò)大傾向,這一過程與氧化應(yīng)激水平及細(xì)胞凋亡增加及炎癥反應(yīng)加重等病理生理變化相關(guān)；2)ASMq可以有效緩解燒傷創(chuàng)面早期進(jìn)行性加深過程中的組織結(jié)構(gòu)損傷和氧化應(yīng)激水平、炎癥反應(yīng)及細(xì)胞凋亡的增加,其作用與劑量相關(guān)；3)ASMq對燒傷創(chuàng)面早期進(jìn)行性加深的治療作用,可能主要通過影響氧化應(yīng)激、Ras/Erk/p90RSK介導(dǎo)的線粒體凋亡途徑及NF-kB介導(dǎo)的炎癥反應(yīng)來實(shí)現(xiàn)。
[Abstract]:Objective: to simulate the pathophysiological process of the early progressive burn wound of two degree burn wound by building a rat model of comb like scald, and to observe the effect of different doses of Abnormal Savda Munziq (ASMq) on the progressive deepening process of burn wound, and explore the potential dose effect and related mechanism. First, the rat model of burn wound was constructed, which was 200 ~ 220g Sprague-Dawley (SD) rats. After pentobarbital sodium anaesthesia, the cupric cupric cuboid (20mm x 10mm) was immersed in 100 centigrade water for 5 minutes. Then the skin of the rat's back after hair removal was kept for 20 seconds, and four wounds were established by interval 5mm, and the gap between the wound and the wound was taken. Part of the wound tissue (20mm x 9mm) was the representative of the stagnation area. The early progressive changes in the burn wound were confirmed by gross and HE staining in different time periods. Then RT-PCR, ELISA, TUNEL staining, immunofluorescence staining and other methods were used to investigate the mechanism of the early progressive deepening of the burn wound from the molecular level, and to find out possible The intervention time points. Secondly, using different doses of ASMq to give the medicine, from the histology and the molecular level, to observe the effect on the early burn wound and its effect on the related damage mechanism. Finally, we use the specific molecular signal inhibitor to identify the signal pathway that may mediate the protective effect of ASMq. Observation under the HE staining microscope, we found that after deep two degree burn, the stasis area of rats was congested and necrotic. It was seen that the two direct burn wounds were observed under the fusion trend and observed under the microscope. Within 72 hours after the burn, the level of oxidative stress in the skin tissue of the burn wound area increased gradually as time changed, and the level of oxidative stress in the skin tissue of the burn wound was gradually increased. The level of GPx (SOD) was significantly lower than that in the normal control group (P0.05). 48 hours after the burn, the level of oxidative stress reached its peak. At the same time, the key enzymes of the two main routes of free radicals - xanthine oxidase (Xanthine Oxidase, XO) and the archetypal nicotinamide adenine dinucleotide phosphate ( Nicotinamide adenine dinucleotide phosphate, NADPH) oxidase 4 (non-phagocytic cell Oxidase 4, Nox4) increased significantly after burn (P0.05). The distribution and release of inflammatory mediators (MPO, IL-1 beta,) in the tissues of the rats after burn increased with time, 48 hours. The expression of activated NF- kappa B increased with time (P0.05), and there was a significant difference between each time group and the normal control group. In addition, the apoptotic cell apoptosis increased obviously in the skin tissue after the burn, accompanied by the activated apoptosis related protein - cysteine - containing aspartate protein hydrolase 3 The expression of /9 (cleaved cysteinyl aspartate specific proteinase, Cleaved Caspase 3/9, CC3/9) was up-regulated. In part second, we observed that ASMq therapy could effectively reduce skin tissue damage in the stagnation area. OD) level (P0.05), and dose-dependent reduction of XO and Nox4 expression (P0.05).ASMq can also relieve the release of inflammatory mediators in the tissues of the stagnant region (P0.05), and increase the activation level of NF- kappa B (P0.05). The effect of high dose ASMq therapy is more obvious, and LPS intervention can reverse the therapeutic effect of high dose. The distribution and expression of CC3/9 also decreased significantly (P0.05), and the effect of ASMq was dose-dependent; Erk activating blocker PD98059 could effectively reverse the effect of high dose ASMq therapy on the apoptosis of tissue cells and down regulation of CC3/9 expression (P0.05). The third part, we investigated the signaling pathways involved in the possible participation of ASMq. The experimental results showed that: 1) ASMq therapy could reduce the activation of NF- kappa B, and the effect of high dose ASMq was more significant, and the highest dose was most obvious (P0.01); 2) ASMq therapy could affect the expression of Bad phosphorylation and Bcl-xL. cytochrome C (Cytochomre C, Cyto). The increase of Bad phosphorylation (P0.01) showed a dose-dependent effect (P0.01). For Bcl-xL, the up regulation of high dose ASMq was more obvious. High dose ASMq therapy could significantly decrease the expression of Cyto C caused by burn, and the high dose effect was more obvious. High dose ASMq on Bad phosphorylation and Bcl-xL, cytochrome C (cytochrome C) expression The effect can be reversed by PD98059, suggesting that Erk activation plays an important role in the role of ASMq; 3) according to the existing literature, ASMq may regulate the cascade of Ras/Erk/p90RSK signals, and this cascade is also an important cascade of modulation signals upstream of the mitochondrial apoptosis signaling pathway. Immunofluorescence staining shows that with the increase of dose, ASM Q treatment could significantly increase the Erk (p-Erk) distribution of phosphorylation, and Western blotting results also showed that low, medium and high dose ASMq could further increase the activation of Erk phosphorylation, with a dose-dependent increase (P0.01), PD98059 could significantly inhibit the activation of ASMq to Erk (P0.01). The effect of the high dose group was more obvious (P0.01), while PD98059 had no effect on the expression of Ras (P0.05). High, middle and low doses of ASMq had a significant up-regulated effect on the expression of p90RSK, corresponding to the activation of Erk phosphorylation, and a dose-dependent effect, while Erk inhibitor PD98059 significantly lowered the p90RSK expression level of the high dose ASMq increase. (1) there is a dynamic progressive deepening process of burn wounds above two degrees in the early stage, and the skin tissue in the vicinity of the initial wound surface has a tendency to expand damage. This process is related to the oxidative stress level, the increase of apoptosis and the aggravation of inflammatory reaction, and 2) ASMq can effectively relieve the early progressive burn wound. The damage of tissue structure and oxidative stress, inflammation and apoptosis are increased in the process, and the effect is related to the dose. 3) the effect of ASMq on the early progressive deepening of burn wound may be realized mainly through the effect of oxidative stress, Ras/Erk/p90RSK mediated apoptosis pathway and NF-kB mediated inflammatory reaction.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R644

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本文編號(hào)：1987014