VAC减轻烧伤瘀滞区坏死的实验研究

发布时间：2018-03-09 20:52

本文选题：封闭负压引流技术　切入点：烧伤　出处：《第四军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：热能可引起烧伤部位即刻而不可逆性的坏死，邻近该部位未直接烧伤部位也可产生损伤，而且损伤程度进行性加重，不断加深扩大，最终导致组织坏死。这种现象的发生不仅加重了患者的病情，而且增加烧伤的致残率及致死率，是烧伤治疗面临的问题之一。封闭负压引流(vacuum-assisted closure，VAC)疗法是利用负压吸引装置与特殊敷料结合后，在创面处产生低于大气压的压力，促进创面愈合的技术，已广泛应用于各类创面的治疗。有研究证明封闭负压引流治疗可逆转烧伤瘀滞区，，减少烧伤创面损伤深度及坏死面积，但缺乏进一步的机制研究。在烧伤瘀滞区进行性坏死过程中，血管内皮细胞既是损伤的主要靶细胞，也是病理进展的效应细胞。有效的保护血管内皮细胞，减轻其损伤及功能失常是逆转烧伤瘀滞区的关键。自噬是指细胞消化自身多余、受损、衰老的蛋白质及细胞器，以维持内环境稳定的细胞机制。本课题以大鼠梳状烧伤模型应用封闭负压引流技术进行治疗，从内皮细胞功能和自噬水平的变化，初步探索VAC技术治疗烧伤瘀滞区的效果和作用机制，为临床治疗提供理论依据。实验共分以下四个部分： 1.建立大鼠铜梳烧伤模型。铜梳梳齿间未直接烧伤的间隙区即为瘀滞区。实验动物随机分为VAC组和对照组。对瘀滞区摄像，用图像分析软件，测量烧伤瘀滞区坏死面积的变化。HE染色观察瘀滞区坏死深度。结果显示：烧伤6小时后VAC组烧伤瘀滞区坏死面积及深度明显小于对照组。 2. NO和ET-1含量检测。我们用硝酸还原酶法检测瘀滞区组织的一氧化氮（NO）的含量，用ELISA法检测内皮素-1(ET-1)水平，对比VAC组和对照组之间两者比值的差异，分析VAC对血管内皮细胞分泌功能的影响。结果显示：烧伤后VAC组瘀滞区组织NO含量明显多于伤前对照组，伤后12小时达到峰值，72小时仍高于正常值。对照组中NO含量低于VAC组，伤后6小时达到峰值，12小时即降低到正常含量以下，并持续下降。两组伤后6小时及其后各时间点差异有统计学差异；烧伤后早期VAC组烧伤区瘀滞组织中ET-1含量较伤前对照组明显上升，伤后24小时达到峰值，48小时后低于正常值。对照组烧伤瘀滞区组织中ET-1含量也上升，但上升程度低于VAC组，伤后6小时达到峰值，伤后12小时即降低到正常值以下。两组伤后6、12、24小时差异有统计学意义（P＜0.05）。伤后早期VAC组ET-1/NO比值较低，12小时到达最低值后逐步回升，接近正常值。而对照组ET-1/NO比值持续升高，在72小时仍很高。两组伤后6小时及其后各时间点差异均有明显差异（P＜0.05）。 3. TM（血栓调节蛋白）和TM mRNA检测。用免疫组化染色和实时荧光PCR检测内皮细胞TM和TM mRNA表达，分析VAC治疗对血管内皮细胞抗凝血、抗炎功能的影响。结果显示：伤后各时间点VAC组瘀滞区组织均可见TM阳性表达。对照组伤后12小时可见TM阳性表达，但伤后24小时及其后时间点均为阴性。伤后各时间点，两组TM mRNA表达均呈下降趋势，但VAC组表达量高于对照组。伤后6小时及其后各时间点差异明显（P＜0.05）。 4. LC3和Beclin-1表达检测。免疫组化染色观察烧伤瘀滞区LC3及Beclin-1的表达，对比VAC组与对照组之间的差异，分析VAC治疗对烧伤瘀滞区细胞自噬的影响。结果显示: VAC组瘀滞区LC3和Beclin-1表达率伤后6至24小时低于对照组，伤后48至72小时高于对照组，半定量分析显示两组在伤后6小时及其后各时间点差异有统计学意义（P＜0.05）。
[Abstract]:Heat can cause burns immediate and irreversible necrosis, adjacent to the site without direct burn can also cause damage, and the damage degree of progressive deepening, expanding, resulting in tissue necrosis. This phenomenon not only aggravate the patient's condition, but also increase the burn rate of morbidity and mortality, is one of the burn treatment problems. VSD (vacuum-assisted closure, VAC) therapy is the use of negative pressure suction device with special dressings combined, produce subatmospheric pressure on the wound, promote wound healing technique has been widely used in the treatment of various wounds. Studies have shown that vacuum sealing drainage treatment can reverse burn stasis stagnation zone, reduce the damage of deep burn wound and necrosis area, but the lack of further studies. In the process of burn necrosis of Stasis Zone, both vascular endothelial cells Is the main target of cell damage, is the pathological progress of effector cells. Vascular endothelial cell protection effectively, reduce the injury and dysfunction is the key to reverse the burn Stasis Zone. Autophagy is refers to the cell to digest their excess, damaged proteins and organelles of aging, to maintain a stable environment within the cellular mechanism of this subject. By using the rat comb burn model of vacuum sealing drainage for treatment, from the changes of endothelial cell function and autophagy, explore the effect and mechanism of VAC technique in the treatment of burn Stasis Zone, provide a theoretical basis for clinical treatment. The experiment is divided into the following four parts:
1. to establish a rat model of burn. Copper comb gap between copper comb comb is not directly burn the Stasis Zone. The experimental animal were randomly divided into VAC group and control group. The camera of the Stasis Zone, using image analysis software to observe the Stasis Zone of necrosis depth changes of.HE measuring burn Stasis Zone of necrosis area staining. The results showed that 6 hours after burn VAC group burn the Stasis Zone of necrosis area and depth significantly less than the control group.
2. the detection of NO and ET-1 content of nitric oxide. We detected by nitrate reductase method the Stasis Zone tissue (NO) were detected by ELISA et -1 (ET-1) level, the difference of the ratio between the two comparison between VAC group and control group, to analyze the effect of VAC on the secretion of vascular endothelial cells. The results showed that burn the content of NO in group VAC was significantly more than the Stasis Zone tissue injury control group before and 12 hours after injury and reached the peak, 72 hours is still higher than normal. NO content of control group was lower than that of VAC group, 6 hours after injury and reached the peak, 12 hours reduced to normal levels, and continued to decline. There were significant differences in 6 hours the two groups after injury and after each time point of difference; early lag in VAC group after burn in burn area and the content of ET-1 is the pre injury control group increased significantly, and reached the peak at 24 hours after injury, 48 hours less than normal. The control group also ET-1 content in blood stasis stagnation area burn The rise, but the rise is less than the VAC group, and reached the peak at 6 hours after injury, 12 hours after injury is reduced to below normal. Two groups of 6,12,24 hours after injury was statistically significant (P < 0.05). Early after injury VAC group ET-1/NO ratio is low, 12 hours to reach the lowest value gradually rise, close to normal value. While the control group, the ratio of ET-1/NO increased in 72 hours is still very high. 6 hours after injury and two groups had significant difference at each time point after the difference (P < 0.05).
3. TM (thrombomodulin). The expression of mRNA and TM detected by immunohistochemical staining and real-time fluorescence PCR detection of endothelial cells TM and TM mRNA analysis of VAC treatment on vascular endothelial cell function of anticoagulant, anti-inflammatory effects. The results showed that the positive expression of TM in the VAC group the Stasis Zone tissue could be seen after injury 12 hours after injury. The control group showed positive expression of TM, but 24 hours after injury and later time points were negative. At each time point, the expression of mRNA TM in two groups were decreased, but the expression of VAC group was higher than the control group. 6 hours after injury and after each time point difference (P < 0.05).
4. LC3 and Beclin-1 expression was detected by immunohistochemical staining. The expression of LC3 and Beclin-1 to observe the burn of the Stasis Zone, the difference between the VAC group and the control group comparison, analysis of effect of VAC treatment on burn cell Stasis Zone of autophagy. The results showed that VAC LC3 and Beclin-1 group the Stasis Zone expression rate after 6 to 24 hours compared with the control group, 48 hours after injury to 72 higher than the control group, semi quantitative analysis showed that the two groups in 6 hours after injury and after each time point had significant difference (P < 0.05).

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R644

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