用伊文思蓝染色观察血管移植过程中血管保存液对内皮的损害

Evans blue dye reflect the impair of endothelium caused by different preserving solutions

目的通过伊文思蓝(Evans blue)染色观察维拉帕米、硝酸甘油混合液(VG液)和盐酸罂粟碱溶液作为血管移植过程中的保存液对内皮层的损害,为临床应用提供依据。方法实验用雄性SD大鼠10只,随机分为2组,分别命名为A组和B组,每组5只。麻醉后SD大鼠迅速摘出胸主动脉在Krebs henseleit buffer(KHB)溶液(4℃)分割,每段长度约1cm,A组中分段后的血管分别被放入4℃的2种由不同公司提供的VG溶液中(分别称为VG1,VG2)、盐酸罂粟碱(1mg/ml)、阴性对照(KHB溶液,pH7.4)以及阳性对照(KHB溶液,pH7.4)中培养30min。B组分段后的血管分别被放入4℃的由2种VG液中不同的成分在林格液中配制后的液体(分别称为PEVG1,MaVG2)、阴性对照(KHB溶液,pH7.4)以及阳性对照(KHB溶液,pH7.4)中培养30min。取出经过上述液体处理后的血管段再经过30min连续充氧的KHB(37℃)液培养,后用Evans blue(0.17%)染色15s。而后浸泡于100μl甲酰胺溶液中过夜,取上清用分光光度计测定Evans blue浓度,再通过蛋白定量测定血管蛋白质含量,得出每微克蛋白质中的Evans blue含量。结果在A组中3种保存液与阳性对照均有差异(P<0.05),2种VG液与罂粟碱溶液有显著性差异(P<0.01)。在B组PEVG 1与对照组有显著性差异(P<0.01)。结论VG液作为血管移植过程中的保存液可以最大限度的减少内皮细胞损伤,相反用盐酸罂粟碱溶液可造成内皮细胞的明显损伤。作为动脉移植过程的保存液VG液具有更安全更有效的优势。

Objective To observe the impair of artery endothelium caused by different vempamil plus nitroglycerin solutions（VG solution）and papaverin solution during dyeing in order to instruct the clinical application. Methods the artery grafting by the method of Evans blue Ten rats were randomly divided into two groups. Thoracic aortar were taken from anaesthetic SD rats and immediately were cut into 1 cm segments in 4℃ KHB. In A group, 5 segments were respectively immersed into two different VG solutions （provided by two different companies）, papaverine solution（ 1 mg/ml ）, control solution（ KHB, pH7.4）for 30min. In B group, 4 segments were respectively incubated in two different VG solution additives （without vempamil, nitroglycerin, heparin and 7% NaHCO3 ） and control solution for 30min. Then all the segments were incubated in KHB for 30min, which was aerated with a gas mixture of 95 % oxygen and 5 % carbon dioxide at 37 ℃. After that, the endothelium of these vessels were dyed with Evans blue （0.17%） for 15 seconds. After keeping the pictures of tinct endothelium by the scanner, these vessels were kept into the formamide （100td） solution overnight. The supematant concentration of Evans blue could be determined by spectrophotometer, then these vessels were immersed into 400μl NaOH （1mol/L） for a week for determination of protein content. Results In A group, the differences between the two VG solutions, papaverine and the positive control were statistically significant（ P 〈 0.05）, between the VG1 ,VG2 and papaverine were remarkablt significant（ P 〈 0.01 ）. In B group the differences between PEVG1 and the positive control were remarkably significant（ P 〈 0.01）.Conclusion The results indicate that using VG solution to prepare for artery bypass grafting maximally preserves endothelium. In contrast, papaverine impairs endothelium. VG solution may be effectively and safely used to prepare for artery bypass grafting.