【作者】 谭家祺；

【导师】 陈绍宗；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【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（P<0.05），创面深度减少最多（P<0.05）。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，P>0.05，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（P<0.05），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（P<0.05），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。更多还原

【Abstract】 Background: Bunrs is a dynamic injury. Burn wound is not permanent, under multiplefactors it will be deeper and wider. The nature of this burn wound progression is death ofcell in the zone of stasis, main causes of cell death include inflammatory cell infiltrates,blood flow changes, and oxygen derived free radicals. Traditionally, death of cell wasdivided into two kinds, apoptosis and oncosis, however, the type II programmed celldeath, autophagy, has not been studied in burn wound yet. Negative pressure woundtherapy (NPWT) has been widely used over diversed acute and chronic wound in peaceand war, and its mechanism has been gradually disclosed either, include edema remove,exudation draining and improve local blood flow. The effect of topical oxygen therapy(TOT) for wound is positive, although it mechanism needs further elucidate. NPWT andTOT have been alone used in burn wound, and our research team member Zhang ZP andLiu HP have proved that combined theses two therapies could improve ischemic woundhealing successfully, but their mechanisms are still not well known. This experimentaimed to build a reliable deep partial thickness burn wound, and explored the cause ofcell death in the zone of stasis, and then used negative pressure combined topical oxygentherapy for burn wound to further elucidate their mechanisms and synergistic effect ofwound treatment and provided evidence for clinical use. This experiment had5parts asbelow. Methods and ResultsPart I: Based on reference, we customed made a brass comb and heated it in boilingwater for5min, blotted dry, and then quickly placed on the shaved and depilated dorsumof rat, and held for12s,16s,20s,24s,28s without any pressure, and then a burn woundwith3interspaces were made. We used image analysis and hematoxylin and eosinstaining to observe the change of interspace area and burn wound depth, and combinedwith gross and microscope observation to analyse. Results: Brass comb burn with20scould make a deep partial thickness burn wound, and interspaces would have burn woundprogression, which is met the standard of the zone of stasis animal model.Part II: Immunohistochemistry was used for analysis the distribution of autophagicmarker Beclin1and apoptotic cell by TUNEL in the zone of stasis. Western blotting wasused for evaluation Bcl-2and Bax, which were dual-regulators of autophagy andapoptosis. Autophagy was mainly strongly expressed in hair follicle epithelium from2hours postburn, and rapidly arrived peak at12hours postburn then gradually and gentlydeclined. Differently, apoptosis was mainly located in stratum epidermis and lightlyexpressed until12hours postburn, and then slowly increased and obvious staining wasobserved at48hours postburn. These results suggest that early treatment for burn woundprogression should take autophagy int consideration.Part III: Negative pressure combined topical oxygen for rat dorsal deep partial thicknessburn wound. There are negative pressure group(NP, continuous negative pressure75mmHg,4hrs), topical oxygen group (TO,40%±5%humidifying pure oxygen,90min),negative pressure combined topical oxygen group (NO, continuous negative pressure75mmHg and40%±5%humidifying pure oxygen at the same time,4hrs), and controlgroup (CT, dressing by single layer oil gause). All is change fresh dressing per two days.Biopsies were taken before treatment, and after treatment at2,4,6and8days. NOgroup’s interspace area decreased the least and burn wound depth declined the most(P<0.05). Except CT group, between the treatments from2d to8d, comparison of otherthree groups’interspaces change, the difference was not statistically significant. Thissuggested that different groups’effect of limiting the zone of stasis progression was mainly in2days.Part VI: Observation of oxidative stress index of rat deep partial thickness burn woundinterspace after four different treatments. The same groups as Part III, samples wereharvested from interspace before treatment and after treatment at2,4and6days, andrelated kits were used to analyse the activity of superoxide dismutase (SOD) and catalase(CAT) and the level of malondialdehyde (MDA). NO group’s activity of SOD and CATwere promoted and level of MDA was lower at each time point, comparison in groups,the statistically difference is significant (P<0.05). NO group could obviously attenuatethe oxidative stress response of deep partial thickness burn wound progression.Part V: The same groups as Part III, samples were harvested from interspace beforetreatment and after treatment for2,4and6days, qRT-PCR was used for semiquantitative NOS, XOD and NOX mRNA expression. NO group’s NOS, XOD and NOXmRNA expression decreased from treatment began(P<0.05), indicated that its protectionof the zone of stasis from oxidative stress was better than other groups.ConclusionBrass comb burn model is a reliable and repeative animal model for burn woundprogression research. After heated brass comb in boiling water for5minutes, and thenplaced it on skin for20seconds could make a deep partial thickness burn wound. Thiswound would go deeper and wider, and unburn skin of interspace would be progressivenecrosis in48hours. Autophagy and apoptosis play roles in burn wound progression, andtheir relative importance would change along with time. Autophagy occured earlier andmainly in dermis layer hair follicle, and apoptosis occurred later and most in epidermis.Negative pressure combined topical oxygen could obviously limit burn woundprogression, considering autophagy occurs in2hours postburn, and its applied timeshould be controlled in2-3hours postburn and maintained for48hours at least. Negativepressure therapy could clear necrotic tissues, edema remove and improve blood flow,these are beneficial for oxygen diffusion. Two therapy combine together could meetmutual complementarity. NO for deep partial thickness burn wound could promote theactivity of SOD and CAT, and decrease the level of MDA, and decline the mRNA expression of NOS, XOD and NOX. Attenuate oxidative stress and limit burn woundprogression, these might be synergetic mechanism of negative pressure combined topicaloxygen.更多还原