负压联合局部给氧治疗大鼠深Ⅱ度烧伤创面进行性坏死的实验研究

发布时间：2018-03-03 14:16

本文选题：负压联合局部给氧创面治疗技术　切入点：负压创面治疗技术　出处：《第四军医大学》2013年博士论文　论文类型：学位论文

【摘要】：背景烧伤是一种动态的损伤。烧伤创面不是完全固定不变，在多种因素的影响下可使创面加深扩大。烧伤创面加深扩大的本质是瘀滞区的细胞死亡，原因有三：炎症细胞的浸润，血流的改变和氧自由基的损伤。传统上认为烧伤瘀滞区细胞死亡有凋亡和胀亡两种，但未见由自噬引起死亡的报道。负压创面治疗技术（Negativepressure wound therapy, NPWT）已广泛应用于平时和战时的各种急慢性创面治疗中，其作用机理也被逐渐揭示，主要是去除水肿，引流渗出液，改善局部血运。近年报道的局部氧疗方法（Topical oxygen therapy，TOT），虽然其作用机制有待深入研究，但文献报道其对创面的治疗效果是肯定的。本室张自鹏和刘海波先后将两者联合应用促进缺血创面愈合的研究，效果明显，但作用机制尚不完全清楚。本实验拟建立深II度烧伤模型，探讨瘀滞区细胞死亡的原因，进一步探索负压和局部给氧在治疗创面的作用机理，为两者联合应用提供实验依据。实验共分以下五个部分： 1.按文献报道用铜梳的突出部分别接触大鼠背部脱毛区12s,16s,20s,24s,28s形成烫伤创面，以烫伤区之间的间隙区作为瘀滞区。利用图像分析和HE染色方法，观察测量间隙区创面面积和烧伤创面深度的变化，结合大体和镜下观察进行分析。结果：铜梳烫伤时间20s可形成深II度烧伤创面，且48h内间隙区会发生进行性坏死，符合瘀滞区动物模型的研究要求。 2.利用免疫组化检测自噬标记物Beclin1和TUNEL法检测瘀滞区组织凋亡细胞，使用蛋白印迹方法检测自噬和凋亡共调节蛋白Bcl-2与Bax的蛋白表达量变化，并探讨两种细胞死亡方式在烧伤创面早期进行性坏死的意义。结果：自噬和凋亡相比，自噬发生较早，2h即出现，12h达到高峰，24h开始下降，主要分布于真皮深层的毛囊；而凋亡出现较晚，12h开始出现，48h才有明显表达，主要分布在表皮层。抗凋亡蛋白Bcl-2的表达量随着时间下降，促凋亡Bax的蛋白表达量随着时间上升。这些结果提示我们对于烧伤创面进行性坏死的早期针对性治疗应将自噬考虑在内。 3.应用负压联合局部氧疗实验性治疗大鼠背部深II度创面。分为单纯负压组（持续负压75mmHg，4小时），单纯氧疗组（单纯给予40%±5%湿化纯氧，90min），负压氧联合组（负压治疗同时给予40%±5%湿化纯氧，4小时）和对照组（单层油纱布+干燥无菌纱布包扎），均隔日换药，分别于治疗前，治疗后2、4、6、8天取材，观察四种治疗对瘀滞区的影响。结果：负压联合给氧组瘀滞区面积下降最少（P0.05），创面深度减少最多（P0.05）。除对照组外，2d-8d的治疗周期内，其余三组面积变化无显著差别，P0.05，说明各治疗组抑制瘀滞区扩大的作用主要在2d内。 4.观察四种治疗方法对大鼠深II度创面瘀滞区组织相关氧化应激指标的影响。同实验3进行分组，分别于治疗前，治疗后2、4、6天取瘀滞区组织，利用相关试剂盒，检测该区组织超氧化物歧化物（SOD）和过氧化物酶（CAT）的活性，丙二醛（MDA）的水平。结果：负压氧联合组各时间点的SOD和CAT活性，MDA水平与其余三组相比较，具有统计学差异（P0.05），该组改善深II度创面的氧化应激反应效果最佳。 5.同上分组治疗深II度创面，分别于治疗前，治疗后2、4、6天取材瘀滞区组织，利用qRT-PCR的方法检测NOS，XOD，NOX三者mRNA表达量。结果：从治疗开始负压氧联合组的NOS，，XOD，NOX mRNA表达量持续下降（P0.05），显示其对瘀滞区抗氧化保护作用优于其他各组。结论：铜梳烫伤模型是一个可靠的可重复的用于研究烧伤进行性坏死的模型。沸水加热5分钟后，铜梳接触皮肤时间控制在20s可形成深II度创面，创面深度进行性加深，未直接烫伤的间隙区皮肤会在48h内损伤进行性坏死。自噬、凋亡均与瘀滞区细胞死亡相关，但发生影响的时间和部位有别。自噬发生较早，主要集中于真皮层毛囊，而凋亡出现较晚，多出现在表皮层。负压联合局部给氧治疗深II度创面可明显抑制创面的加深和扩大。由于自噬的发生时间在伤后2h，故治疗时间理论上应不晚于2-3h，且至少维持48h。负压治疗能够清除坏死组织、减轻水肿和改善血运，氧疗刚直接向创面弥散氧，两者联合使SOD, CAT活性提高，MDA含量降低，使ROS相关的NOS, XOD, NOX的mRNA表达量降低，降低氧化应激反应，限制瘀滞区进行性坏死，这可能是负压联合局部给氧疗法的协同作用机制之一。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R644

【参考文献】