负压封闭引流术减轻兔骨骼肌缺血再灌注损伤的作用机制研究  

Mechanism of vacuum sealing drainage therapy attenuating ischemia-reperfusion injury of skeletal muscle in rabbit

作　　者：王翔[1] 杨帆[1] 管震[1] 王东方[1] 白祥军[1] 高伟[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院创伤外科,武汉430030

出　　处：《中华外科杂志》2016年第4期292-296,共5页Chinese Journal of Surgery

基　　金：湖北省科技计划基金资助项目（2015CFB670）

摘　　要：目的 探讨负压封闭引流（VSD）技术减轻兔骨骼肌缺血再灌注损伤（I/R）的相关作用机制.方法 30只新西兰大耳兔随机分成对照（假手术）组、I/R组和I/R＋ VSD组,每组10只.通过阻断4h和再灌注6h左后肢股动脉和静脉的方法建立I/R模型,在此模型基础上使用VSD技术进行再灌注时的干预即为I/R＋ VSD组.对各组骨骼肌组织进行髓过氧化物酶（MPO）、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽（GSH）的测定.HE组织切片观察细胞水肿程度;免疫组化评估骨骼肌细胞表达高迁移率族蛋白B1（HMGB1）的情况,并采用相对实时定量PCR和免疫印迹法分别在mRNA水平、蛋白表达水平对各组骨骼肌细胞HMGB1的变化程度进行分析.所得数据采用方差分析进行检验.结果 I/R组MPO（0.91 ±0.22）、MDA（2.04 ±0.92）、SOD（35.97±9.23）、CAT（31.42±16.27）、GSH（1.48±0.90）的组织含量与VSD干预组（0.53 ±0.08、1.65±1.02、55.99±18.97、48.50±17.86、3.54±1.88）和对照组（0.31 ±0.10、1.01±0.12、61.83±14.91、75.95±13.09、3.84 ±2.08）比较,差异有统计学意义（F=26.480、4.250、5.240、9.720、5.240,P =0.000、0.040、0.020、0.002、0.020）.HE染色结果显示,I/R组骨骼肌细胞的间隙比高于VSD干预组和正常对照组（F=16.47,P＜0.05）;免疫组化检测结果表明,对照组、I/R组和I/R＋ VSD组HMGB1阳性细胞百分比分别为1.94％、18.63％和61.36％,三组间差异有统计学意义（F=853.886,P＜0.01）,此结果与实时定量PCR（F=50.653,P＜0.01）和免疫印迹法（F=963.489,P＜0.01）检测结果相符.结论 VSD技术可通过增加骨骼肌细胞的抗氧化应激能力、降低氧化因子损伤和炎性介质因子的表达而减轻缺血后再灌注时的损伤程度.Objective To investigate the mechanism of how vacuum sealing drainage （VSD） ameliorating ischemia reperfusion （I/R） injury in skeletal muscle I/R model.Methods Thirty New Zealand white rabbits were divided into three groups：control （sham operation） group,I/R group,VSD ＋ I/R group.The ischemia of the left hind limb of the animal was induced by clamping the common femoral artery and vein.After 4 hours of ischemia,the clamp was removed and the hind limp underwent 6 hours reperfusion.VSD treated animals received the treatment at the beginning of reperfusion.The concentrations of myeloperoxidase（MPO）,malondialdehyde （MDA）,superoxide dismutase （SOD）,catalase （CAT） and glutathione（GSH） in muscular tissues were assayed.HE stained pathological section was used to evaluate the degree of edema of muscular tissues,and the immunohistochemistry was used to detect the percentage of positive cells expressing high mobility group protein B1 （HMGB1）.Q-RT-PCR and Western Blot were used to detect the mRNA levels and protein expression of HMGB1 in myocyte respectively.The experimental data was tested using variance analysis.Results The levels of inflammatory factors and antioxidant factors in muscular tissues were significantly different in the I/R group compared to the VSD group and control group （the levels of MPO in I/R group,I/R ＋ VSD group and control group were 0.91 ± 0.22,0.53 ± 0.08,0.31 ± 0.10,respectively,F =26.48,P =0.000;MDA were 2.04 ± 0.92,1.65 ± 1.02,1.01 ± 0.12,F =4.250,P=0.040;SOD were 35.97 ±9.23,55.99 ± 18.97,61.83 ± 14.91,F=5.240,P =0.020;CAT were 31.42 ± 16.27,48.50 ± 17.86,75.95 ± 13.09,F =9.720,P =0.002;GSH were 1.48 ± 0.90,3.54 ± 1.88,3.84 ± 2.08,F =5.240,P =0.020）.HE staining showed an increased intercellular space ratio in the I/R group （F =16.47,P 〈 0.05）.Immunohistochemistry staining showed that percentage of HMGB1 positive myocytes in control,I/R and I/R ＋ VSD group are 1.94%,18.63% and 61.36%,respectively.There was significant difference

关 键 词：再灌注损伤 肌 骨骼 负压伤口疗法 HMGB1蛋白 

分 类 号：R68[医药卫生—骨科学]