本文選題：負(fù)壓聯(lián)合局部給氧創(chuàng)面治療技術(shù)　切入點：負(fù)壓創(chuàng)面治療技術(shù)　出處：《第四軍醫(yī)大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：背景燒傷是一種動態(tài)的損傷。燒傷創(chuàng)面不是完全固定不變，在多種因素的影響下可使創(chuàng)面加深擴大。燒傷創(chuàng)面加深擴大的本質(zhì)是瘀滯區(qū)的細(xì)胞死亡，原因有三：炎癥細(xì)胞的浸潤，血流的改變和氧自由基的損傷。傳統(tǒng)上認(rèn)為燒傷瘀滯區(qū)細(xì)胞死亡有凋亡和脹亡兩種，但未見由自噬引起死亡的報道。負(fù)壓創(chuàng)面治療技術(shù)（Negativepressure wound therapy, NPWT）已廣泛應(yīng)用于平時和戰(zhàn)時的各種急慢性創(chuàng)面治療中，其作用機理也被逐漸揭示，主要是去除水腫，引流滲出液，改善局部血運。近年報道的局部氧療方法（Topical oxygen therapy，TOT），雖然其作用機制有待深入研究，但文獻(xiàn)報道其對創(chuàng)面的治療效果是肯定的。本室張自鵬和劉海波先后將兩者聯(lián)合應(yīng)用促進(jìn)缺血創(chuàng)面愈合的研究，效果明顯，但作用機制尚不完全清楚。本實驗擬建立深I(lǐng)I度燒傷模型，探討?zhàn)鰷䥇^(qū)細(xì)胞死亡的原因，進(jìn)一步探索負(fù)壓和局部給氧在治療創(chuàng)面的作用機理，為兩者聯(lián)合應(yīng)用提供實驗依據(jù)。實驗共分以下五個部分： 1.按文獻(xiàn)報道用銅梳的突出部分別接觸大鼠背部脫毛區(qū)12s,16s,20s,24s,28s形成燙傷創(chuàng)面，以燙傷區(qū)之間的間隙區(qū)作為瘀滯區(qū)。利用圖像分析和HE染色方法，觀察測量間隙區(qū)創(chuàng)面面積和燒傷創(chuàng)面深度的變化，結(jié)合大體和鏡下觀察進(jìn)行分析。結(jié)果：銅梳燙傷時間20s可形成深I(lǐng)I度燒傷創(chuàng)面，且48h內(nèi)間隙區(qū)會發(fā)生進(jìn)行性壞死，符合瘀滯區(qū)動物模型的研究要求。 2.利用免疫組化檢測自噬標(biāo)記物Beclin1和TUNEL法檢測瘀滯區(qū)組織凋亡細(xì)胞，使用蛋白印跡方法檢測自噬和凋亡共調(diào)節(jié)蛋白Bcl-2與Bax的蛋白表達(dá)量變化，并探討兩種細(xì)胞死亡方式在燒傷創(chuàng)面早期進(jìn)行性壞死的意義。結(jié)果：自噬和凋亡相比，自噬發(fā)生較早，2h即出現(xiàn)，12h達(dá)到高峰，24h開始下降，主要分布于真皮深層的毛囊；而凋亡出現(xiàn)較晚，12h開始出現(xiàn)，48h才有明顯表達(dá)，主要分布在表皮層�？沟蛲龅鞍譈cl-2的表達(dá)量隨著時間下降，促凋亡Bax的蛋白表達(dá)量隨著時間上升。這些結(jié)果提示我們對于燒傷創(chuàng)面進(jìn)行性壞死的早期針對性治療應(yīng)將自噬考慮在內(nèi)。 3.應(yīng)用負(fù)壓聯(lián)合局部氧療實驗性治療大鼠背部深I(lǐng)I度創(chuàng)面。分為單純負(fù)壓組（持續(xù)負(fù)壓75mmHg，4小時），單純氧療組（單純給予40%±5%濕化純氧，90min），負(fù)壓氧聯(lián)合組（負(fù)壓治療同時給予40%±5%濕化純氧，4小時）和對照組（單層油紗布+干燥無菌紗布包扎），均隔日換藥，分別于治療前，治療后2、4、6、8天取材，觀察四種治療對瘀滯區(qū)的影響。結(jié)果：負(fù)壓聯(lián)合給氧組瘀滯區(qū)面積下降最少（P0.05），創(chuàng)面深度減少最多（P0.05）。除對照組外，2d-8d的治療周期內(nèi)，其余三組面積變化無顯著差別，P0.05，說明各治療組抑制瘀滯區(qū)擴大的作用主要在2d內(nèi)。 4.觀察四種治療方法對大鼠深I(lǐng)I度創(chuàng)面瘀滯區(qū)組織相關(guān)氧化應(yīng)激指標(biāo)的影響。同實驗3進(jìn)行分組，分別于治療前，治療后2、4、6天取瘀滯區(qū)組織，利用相關(guān)試劑盒，檢測該區(qū)組織超氧化物歧化物（SOD）和過氧化物酶（CAT）的活性，丙二醛（MDA）的水平。結(jié)果：負(fù)壓氧聯(lián)合組各時間點的SOD和CAT活性，MDA水平與其余三組相比較，具有統(tǒng)計學(xué)差異（P0.05），該組改善深I(lǐng)I度創(chuàng)面的氧化應(yīng)激反應(yīng)效果最佳。 5.同上分組治療深I(lǐng)I度創(chuàng)面，分別于治療前，治療后2、4、6天取材瘀滯區(qū)組織，利用qRT-PCR的方法檢測NOS，XOD，NOX三者mRNA表達(dá)量。結(jié)果：從治療開始負(fù)壓氧聯(lián)合組的NOS，，XOD，NOX mRNA表達(dá)量持續(xù)下降（P0.05），顯示其對瘀滯區(qū)抗氧化保護作用優(yōu)于其他各組。 結(jié)論： 銅梳燙傷模型是一個可靠的可重復(fù)的用于研究燒傷進(jìn)行性壞死的模型。沸水加熱5分鐘后，銅梳接觸皮膚時間控制在20s可形成深I(lǐng)I度創(chuàng)面，創(chuàng)面深度進(jìn)行性加深，未直接燙傷的間隙區(qū)皮膚會在48h內(nèi)損傷進(jìn)行性壞死。自噬、凋亡均與瘀滯區(qū)細(xì)胞死亡相關(guān)，但發(fā)生影響的時間和部位有別。自噬發(fā)生較早，主要集中于真皮層毛囊，而凋亡出現(xiàn)較晚，多出現(xiàn)在表皮層。負(fù)壓聯(lián)合局部給氧治療深I(lǐng)I度創(chuàng)面可明顯抑制創(chuàng)面的加深和擴大。由于自噬的發(fā)生時間在傷后2h，故治療時間理論上應(yīng)不晚于2-3h，且至少維持48h。負(fù)壓治療能夠清除壞死組織、減輕水腫和改善血運，氧療剛直接向創(chuàng)面彌散氧，兩者聯(lián)合使SOD, CAT活性提高，MDA含量降低，使ROS相關(guān)的NOS, XOD, NOX的mRNA表達(dá)量降低，降低氧化應(yīng)激反應(yīng)，限制瘀滯區(qū)進(jìn)行性壞死，這可能是負(fù)壓聯(lián)合局部給氧療法的協(xié)同作用機制之一。
[Abstract]:The background is a dynamic burn injury. The burn wound is not completely fixed, under the influence of various factors can make the wound deepening expand. Burn expansion is the essence of the Stasis Zone of cell death, there are three reasons: the infiltration of inflammatory cells, the changes of blood flow and oxygen free radical damage on the traditional. That burns the Stasis Zone of cell death apoptosis and oncosis in two, but no reports of death by autophagy. Negative pressure wound therapy (Negativepressure wound, therapy, NPWT) has been widely used in peacetime and wartime a variety of acute and chronic wound treatment, the mechanism is also gradually revealed, is mainly to remove edema. Drainage of exudate, improve local blood circulation. The local oxygen therapy methods reported in recent years (Topical oxygen therapy, TOT), although the mechanism remains to be investigated, but reported on wound treatment effect is positive. The Zhang Zipeng and Liu Haibo will have to promote research and the application of the combination of ischemic wound healing effect is obvious, but the mechanism is not fully understood. This study intends to establish a deep II degree burn model, to explore the causes of the Stasis Zone of cell death, to further explore the negative pressure and local oxygen therapy in treatment of the wound mechanism, to provide experimental basis for for the combination of the two. The experiment is divided into the following five parts:
1. according to the reported projections with copper comb don't contact with 12s, back hair removal zone of rat 16S, 20s, 24s, 28s formed the scald wound, with the gap between the area of the burn area as the Stasis Zone. By using image analysis and HE staining method to observe the changes of wound gap measured area and deep burn wound degree. With gross and microscopic observation were analyzed. Results: the copper comb burn time 20s can form deep II degree burn wound, and the 48h in gap region occurs progressive necrosis of Stasis Zone, conform to the study of animal model.
2. immunohistochemistry staining was used to detect autophagy markers Beclin1 and TUNEL were used to detect the Stasis Zone tissue cell apoptosis, expression of autophagy and apoptosis using Western blot method to detect the total regulatory protein Bcl-2 and Bax protein, and to explore the two kinds of cell death by necrosis in early burn wound. Results: compared the significance of autophagy and apoptosis autophagy occurs earlier, namely, 2h, 12h peak, 24h began to decline, mainly distributed in the deep dermis of the hair follicle; apoptosis appeared later, 12h began to appear, 48h have obvious expression, mainly distributed in the epidermis. The expression of anti apoptosis protein Bcl-2 decreased with time, the pro apoptotic Bax protein expression as the time increased. These results suggest that our early necrosis for burn wound treatment should be targeted autophagy into account.
3. application of negative pressure combined with local oxygen therapy in treatment of experimental rat back deep II degree burn wounds. Divided into simple negative pressure group (continuous negative pressure 75mmHg, 4 hours), pure oxygen therapy group (treated with 40% + 5% humidification oxygen, 90min), negative pressure oxygen combined group (negative pressure treatment with 40% + 5% oxygen humidification, 4 hours) and control group (single oil gauze + dry sterile gauze), were respectively in the dressing was changed every day, before treatment, after treatment 2,4,6,8 days, to observe the effects of four kinds of treatment of Stasis Zone. Results: negative pressure combined to the area of stagnation area blood stasis group decreased oxygen minimum (P0.05), reduce the depth of the wound most (P0.05). Except for the control group, 2d-8d treatment period, no significant difference between the three groups, the area change of P0.05, shows that the treatment group inhibited the Stasis Zone expanding role mainly in 2D.
4. to observe the effects of four kinds of treatment methods on rat deep II degree Wound Stasis Zone tissue oxidative stress index. In the same experiment 3 groups respectively before treatment, the Stasis Zone tissue 2,4,6 days after treatment, the use of the kit, the detection of tissue superoxide dismutase (SOD) and peroxide the enzyme (CAT) activity, malondialdehyde (MDA) level. Results: the combination of SOD and CAT activity of each time point group of negative pressure oxygen, MDA levels compared with the other three groups, with statistical difference (P0.05), the group improved the oxidative stress effect of deep II degree burn wounds is the best.
5.. Grouping treatment of deep II degree burn wounds, respectively before treatment, the Stasis Zone tissue 2,4,6 days after treatment, the use of qRT-PCR method for detection of NOS, XOD, NOX three mRNA expression. Results: from the start of treatment of negative pressure oxygen combined with group NOS, XOD, NOX mRNA expression decreased continuously (P0.05). The display of the Stasis Zone of antioxidant effect than other groups.
Conclusion:
Copper comb scald model is a reliable and reproducible study for burn progressive necrosis model. The boiling water after 5 minutes of heating, the copper comb skin contact time to control the formation of deep II degree burn wound depth in 20s, to deepen, not directly burn the skin gap within the 48h damage of necrosis. Autophagy, apoptosis was associated with cell death of the Stasis Zone, but the time and place of the effects are different. Autophagy occurred earlier, mainly in the hair follicle and dermis, apoptosis appeared later and appear in the epidermis. Negative pressure combined with topical oxygen therapy in deep II degree deepen and expand the wound surface can obviously inhibit the wound. Because time of occurrence of autophagy in 2h after injury, so the treatment time in theory should not later than 2-3H, and at least 48h. negative pressure treatment can remove the necrotic tissue, alleviate edema and improve blood circulation, oxygen therapy just directly to the wound two oxygen diffusion, combined with the SO D, CAT activity increased, MDA content decreased, ROS related NOS, XOD and NOX mRNA expression decreased, decreased oxidative stress and restricted progressive necrosis in stasis area, which may be one of the synergistic mechanisms of negative pressure combined with local oxygen therapy.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R644

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