Tempol对大鼠背部超长任意皮瓣存活影响的实验研究

发布时间：2018-12-12 15:12

【摘要】：实验目的:研究新型小分子氧自由清除剂Tempol对大鼠背部超长任意皮瓣存活的影响,并从抗氧化应激、促进血管生成,减轻炎症反应三个方面探讨其作用机制,为阐明缺血再灌注损伤造成皮瓣远端发生坏死的发生机制提供了理论依据,为如何减轻缺血再灌注损伤造成皮瓣远端出现严重坏死,提高任意皮瓣应用效率,覆盖关键创面提供新思路。实验方法:1.随机将84只雄性成年SD大鼠分为生理盐水对照组和Tempol组,42只/组,所有皮瓣均参考Mcfarlane皮瓣进行切取,于大鼠背部制备面积为9cm*3cm大小的LRPSF。Tempol组术前15min至术后第7天,每天按(100mg/Kg)腹腔注射Tempol(1:10生理盐水稀释后),生理盐水对照组:按相同的方法注射等量的生理盐水。皮瓣移植后及时观察、记录每组实验动物成活及皮瓣存活情况;第7天,测量各组大鼠皮瓣成活率,(1g/ml)明胶氧化铅灌注后行Micro CT血管三维成像观察血管生成情况并利用3D-viewer及MAT-LAB软件计算血管长度和血管容积。2.术后第7天剪取皮瓣中段组织行HE染色观察皮瓣组织层次结构、血管生成、炎症反应及组织坏死程度,同时行免疫组织化学染色观察VEGF表达情况。3.术后即刻及第1,3,7天,将皮瓣中段组织匀浆,采用水溶性四氮唑-1法及可见光法分别测定抗氧化酶SOD和脂质过氧化产物MDA含量;利用ELISA相关试剂盒检测皮瓣组织中炎性因子TNF-α和IL-6含量。实验结果1.所有实验动物均成活至实验完成,各组皮瓣均无感染、皮瓣下血肿皮及瓣下积液。随时间延长,两组皮瓣远端均发生不同程度的坏死,但Tempol组皮瓣坏死程度较生理盐水对照组轻;且Tempol组血管分布及连续性均优于生理盐水对照组。术后第7天Tempol组皮瓣成活率、血管容积及血管总长度均显著高于生理盐水对照组(p0.05或p0.01)。2.术后第7天组织学及免疫组织化学染色观察示,与生理盐水对照组比较,Tempol组皮瓣各层组织结构清楚,微小血管生成明显增多,组织炎症反应及炎性细胞浸润较轻,真皮组织内VEGF蛋白染色程度明显增高。3.测量两组皮瓣组织匀浆后发现,术后1,3,7天两组皮瓣组织SOD、MDA、TNF-α、IL-6差别具有统计学意义,Tempol组MDA、TNF-α、IL-6含量明显低于生理盐水对照组,SOD含量明显高于生理盐水对照组(P0.05);而术后即刻两组皮瓣组织匀浆各项检测指标差异无统计学意义(P0.05)。实验结论:1.Tempol能够显著提高任意皮瓣成活率,减轻皮瓣组织水肿及炎症反应,促进皮瓣存活;2.Tempol能够增加皮瓣组织中VEGF的表达,促进血管生成,从而提高皮瓣成活率;3.Tempol能够显著提高皮瓣组织中SOD含量,降低MDA、TNF-α及IL-6含量,清除氧自由基及抑制炎症因子释放,减轻皮瓣缺血再灌注损伤,从而提高皮瓣成活率。
[Abstract]:Objective: to study the effect of a new small molecular oxygen free scavenger (Tempol) on the survival of overlong random skin flap in rats, and to explore its mechanism from three aspects: antioxidation stress, promoting angiogenesis and alleviating inflammatory reaction. In order to elucidate the mechanism of necrosis of the distal end of the flap caused by ischemia-reperfusion injury, this paper provides a theoretical basis for how to alleviate the severe necrosis of the distal end of the flap caused by ischemia-reperfusion injury, and to improve the efficiency of the application of arbitrary flap. Covering critical wounds provides new ideas. Experimental method: 1. Eighty-four male adult SD rats were randomly divided into saline control group and Tempol group, 42 rats in each group. All the flaps were removed with reference to Mcfarlane flaps. 15min was prepared in the LRPSF.Tempol group with the size of 9cm*3cm before operation until the 7th day after operation. Daily Tempol was injected intraperitoneally by 100mg/Kg (after 1:10 saline dilution) and saline control group was injected with the same amount of saline in the same way. The survival of each group of experimental animals and the survival of the flap were recorded. On the 7th day, the survival rate of the flap was measured. After 1g/ml was perfused with lead oxide gelatin, the angiogenesis was observed by Micro CT angiography, and the vascular length and volume were calculated by 3D-viewer and MAT-LAB software. On the 7th day after operation, HE staining was performed to observe the tissue structure, angiogenesis, inflammatory reaction and degree of tissue necrosis in the middle segment of the flap. Meanwhile, the expression of VEGF was observed by immunohistochemical staining. Immediately after operation and 7 days after operation, the tissue homogenate of the middle segment of the flap was homogenized. The contents of antioxidant enzyme SOD and lipid peroxidation product MDA were determined by water-soluble tetrazole-1 method and visible light method respectively. The content of inflammatory factor TNF- 伪 and IL-6 in skin flap tissue was detected by ELISA related kit. Experimental results 1. All the experimental animals survived until the experiment was completed. There was no infection in all the flaps, and the skin of subcutaneous hematoma and subvalvular effusion. With the extension of time, the distal flap necrosis occurred in both groups, but the degree of flap necrosis in Tempol group was lighter than that in saline group, and the vascular distribution and continuity in Tempol group was better than that in saline control group. On the 7th day after operation, the survival rate, vascular volume and total vascular length in Tempol group were significantly higher than those in saline control group (p0.05 or p0.01). Histological and immunohistochemical staining on the 7th day after operation showed that compared with the saline control group, the skin flap in Tempol group had clear tissue structure, increased microangiogenesis, and slight inflammatory reaction and inflammatory cell infiltration. The staining degree of VEGF protein in dermal tissue was significantly increased by 0.3%. After measuring the tissue homogenate of the two groups, it was found that there was significant difference in SOD,MDA,TNF- 伪 and IL-6 between the two groups on the 1st and 7th day after operation, and the contents of MDA,TNF- 伪 and IL-6 in the Tempol group were significantly lower than those in the normal saline control group. The content of SOD was significantly higher than that of normal saline control group (P0.05). But immediately after operation, there was no significant difference between the two groups in the detection of tissue homogenate (P0.05). Conclusion: 1.Tempol can significantly increase the survival rate of arbitrary flaps, alleviate the edema and inflammatory reaction of flaps, and promote the survival of flaps, while 2.Tempol can increase the expression of VEGF in the flap tissues and promote angiogenesis, thereby increasing the survival rate of the flaps. 3.Tempol could significantly increase the content of SOD, decrease the contents of MDA,TNF- 伪 and IL-6, scavenge oxygen free radicals, inhibit the release of inflammatory factors, alleviate the ischemia reperfusion injury of the flap, and improve the survival rate of the flap.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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