Tempol對大鼠背部超長任意皮瓣存活影響的實驗研究
發(fā)布時間：2018-12-12 15:12
【摘要】：實驗?zāi)康?研究新型小分子氧自由清除劑Tempol對大鼠背部超長任意皮瓣存活的影響,并從抗氧化應(yīng)激、促進血管生成,減輕炎癥反應(yīng)三個方面探討其作用機制,為闡明缺血再灌注損傷造成皮瓣遠端發(fā)生壞死的發(fā)生機制提供了理論依據(jù),為如何減輕缺血再灌注損傷造成皮瓣遠端出現(xiàn)嚴重壞死,提高任意皮瓣應(yīng)用效率,覆蓋關(guān)鍵創(chuàng)面提供新思路。實驗方法:1.隨機將84只雄性成年SD大鼠分為生理鹽水對照組和Tempol組,42只/組,所有皮瓣均參考Mcfarlane皮瓣進行切取,于大鼠背部制備面積為9cm*3cm大小的LRPSF。Tempol組術(shù)前15min至術(shù)后第7天,每天按(100mg/Kg)腹腔注射Tempol(1:10生理鹽水稀釋后),生理鹽水對照組:按相同的方法注射等量的生理鹽水。皮瓣移植后及時觀察、記錄每組實驗動物成活及皮瓣存活情況;第7天,測量各組大鼠皮瓣成活率,(1g/ml)明膠氧化鉛灌注后行Micro CT血管三維成像觀察血管生成情況并利用3D-viewer及MAT-LAB軟件計算血管長度和血管容積。2.術(shù)后第7天剪取皮瓣中段組織行HE染色觀察皮瓣組織層次結(jié)構(gòu)、血管生成、炎癥反應(yīng)及組織壞死程度,同時行免疫組織化學(xué)染色觀察VEGF表達情況。3.術(shù)后即刻及第1,3,7天,將皮瓣中段組織勻漿,采用水溶性四氮唑-1法及可見光法分別測定抗氧化酶SOD和脂質(zhì)過氧化產(chǎn)物MDA含量;利用ELISA相關(guān)試劑盒檢測皮瓣組織中炎性因子TNF-α和IL-6含量。實驗結(jié)果1.所有實驗動物均成活至實驗完成,各組皮瓣均無感染、皮瓣下血腫皮及瓣下積液。隨時間延長,兩組皮瓣遠端均發(fā)生不同程度的壞死,但Tempol組皮瓣壞死程度較生理鹽水對照組輕;且Tempol組血管分布及連續(xù)性均優(yōu)于生理鹽水對照組。術(shù)后第7天Tempol組皮瓣成活率、血管容積及血管總長度均顯著高于生理鹽水對照組(p0.05或p0.01)。2.術(shù)后第7天組織學(xué)及免疫組織化學(xué)染色觀察示,與生理鹽水對照組比較,Tempol組皮瓣各層組織結(jié)構(gòu)清楚,微小血管生成明顯增多,組織炎癥反應(yīng)及炎性細胞浸潤較輕,真皮組織內(nèi)VEGF蛋白染色程度明顯增高。3.測量兩組皮瓣組織勻漿后發(fā)現(xiàn),術(shù)后1,3,7天兩組皮瓣組織SOD、MDA、TNF-α、IL-6差別具有統(tǒng)計學(xué)意義,Tempol組MDA、TNF-α、IL-6含量明顯低于生理鹽水對照組,SOD含量明顯高于生理鹽水對照組(P0.05);而術(shù)后即刻兩組皮瓣組織勻漿各項檢測指標差異無統(tǒng)計學(xué)意義(P0.05)。實驗結(jié)論:1.Tempol能夠顯著提高任意皮瓣成活率,減輕皮瓣組織水腫及炎癥反應(yīng),促進皮瓣存活;2.Tempol能夠增加皮瓣組織中VEGF的表達,促進血管生成,從而提高皮瓣成活率;3.Tempol能夠顯著提高皮瓣組織中SOD含量,降低MDA、TNF-α及IL-6含量,清除氧自由基及抑制炎癥因子釋放,減輕皮瓣缺血再灌注損傷,從而提高皮瓣成活率。
[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- 偽 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- 偽 and IL-6 between the two groups on the 1st and 7th day after operation, and the contents of MDA,TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622
本文編號：2374797
[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- 偽 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- 偽 and IL-6 between the two groups on the 1st and 7th day after operation, and the contents of MDA,TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622
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