国产猪胰弹性蛋白酶联合氯化钙浸泡法构建兔腹主动脉瘤模型
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摘要
目的采用国产猪胰弹性蛋白酶联合氯化钙浸泡法构建简便稳定的兔腹主动脉瘤(AAA)模型。方法 48只新西兰大白兔随机分为动脉瘤组、蛋白酶组、氯化钙组和假手术组。剖腹手术游离一段长约1 cm腹主动脉,动脉瘤组游离动脉段以氯化钙和国产猪胰弹性蛋白酶浸泡20 min,蛋白酶组、氯化钙组分别仅以猪胰弹性蛋白酶、氯化钙浸泡20 min,假手术组由0.9%氯化钠溶液浸泡20 min。术后5、15、30 d采用彩色超声检测浸泡段腹主动脉直径,术后5、30 d每组分别处死6只实验兔作组织病理学和免疫组织化学检查。结果动脉瘤组实验兔均形成AAA,蛋白酶组仅形成1例,氯化钙组、假手术组均未见形成。动脉瘤组与其余各组组织病理学比较,动脉内膜和中膜较薄,胶原纤维和弹力纤维含量减少且修复增生不明显,基质金属蛋白酶(MMP)-2、MMP-9表达和炎性细胞浸润均明显增强。术后5 d蛋白酶组、氯化钙组可见血管破坏,术后30 d蛋白酶组与动脉瘤组相比可见内膜增生明显,中膜厚度恢复至正常水平,胶原纤维、弹力纤维和平滑肌细胞含量增多明显,MMP-2、MMP-9表达和炎性细胞浸润明显减弱。结论国产猪胰弹性蛋白酶联合氯化钙浸泡法可构建简便稳定的兔AAA模型。
Objective To establish a simple and stable rabbit model of abdominal aortic aneurysm(AAA) by using domestic porcine elastase combined with calcium chloride immersion method. Methods A total of 48 New Zealand white rabbits were randomly and equally divided into four groups with 12 rabbits in each group, including aneurysm group, porcine elastase group, calcium chloride group and sham-operation group. Laparotomy was performed for each rabbit, a abdominal aorta about 1 cm long was separated. The separated segment was immersed in the solution of calcium chloride and domestic porcine pancreatic elastase for 20 min(aneurysm group), in the solution of domestic porcine pancreatic elastase for 20 min(porcine elastase group), in the solution of calcium chloride for 20 min(calcium chloride group), and in 0.9% sodium chloride solution for 20 min(sham-operation group) respectively. At 5, 15 and 30 days after treatment the diameter of immersed segment of abdominal aorta was measured with color ultrasound. Every 6 rabbits from each group were sacrificed each time at 5 days and 30 days after treatment, and the specimens were sent for histopathological and immunohistochemical examinations. Results Successful modeling of AAA was obtained in all experimental rabbits of aneurysm group, only one rabbit of porcine elastase group developed AAA, and no formation of AAA was found in both calcium chloride group and sham-operation group. Pathologically compared with other three groups, in aneurysm group the arterial intima and media were thinner, the contents of collagenous fiber and el astic fiber were decreased with less sings of repair and proliferation, the expressions of matrix metallo-proteinase-2(MMP-2) and MMP-9 as well as the degree of inflammatory cell infiltration were all significantly enhanced. Five days after the treatment, vascular destruction was detected in porcine elastase group and calcium chloride group; and 30 days after the treatment in porcine elastase group and aneurysm group obvious intimal hyperplasia was observed, the thickness of tunica media returned to normal level, the contents of collagenous fiber, elastic fibers and smooth muscle cells were markedly increased, while the expressions of MMP-2 and MMP-9 as well as the degree of inflammatory cell infiltration became obviously weakened. Conclusion A simple and stable rabbit model of AAA can be successfully established by using domestic porcine elastase combined with calcium chloride immersion method.
Objective To establish a simple and stable rabbit model of abdominal aortic aneurysm(AAA) by using domestic porcine elastase combined with calcium chloride immersion method. Methods A total of 48 New Zealand white rabbits were randomly and equally divided into four groups with 12 rabbits in each group, including aneurysm group, porcine elastase group, calcium chloride group and sham-operation group. Laparotomy was performed for each rabbit, a abdominal aorta about 1 cm long was separated. The separated segment was immersed in the solution of calcium chloride and domestic porcine pancreatic elastase for 20 min(aneurysm group), in the solution of domestic porcine pancreatic elastase for 20 min(porcine elastase group), in the solution of calcium chloride for 20 min(calcium chloride group), and in 0.9% sodium chloride solution for 20 min(sham-operation group) respectively. At 5, 15 and 30 days after treatment the diameter of immersed segment of abdominal aorta was measured with color ultrasound. Every 6 rabbits from each group were sacrificed each time at 5 days and 30 days after treatment, and the specimens were sent for histopathological and immunohistochemical examinations. Results Successful modeling of AAA was obtained in all experimental rabbits of aneurysm group, only one rabbit of porcine elastase group developed AAA, and no formation of AAA was found in both calcium chloride group and sham-operation group. Pathologically compared with other three groups, in aneurysm group the arterial intima and media were thinner, the contents of collagenous fiber and el astic fiber were decreased with less sings of repair and proliferation, the expressions of matrix metallo-proteinase-2(MMP-2) and MMP-9 as well as the degree of inflammatory cell infiltration were all significantly enhanced. Five days after the treatment, vascular destruction was detected in porcine elastase group and calcium chloride group; and 30 days after the treatment in porcine elastase group and aneurysm group obvious intimal hyperplasia was observed, the thickness of tunica media returned to normal level, the contents of collagenous fiber, elastic fibers and smooth muscle cells were markedly increased, while the expressions of MMP-2 and MMP-9 as well as the degree of inflammatory cell infiltration became obviously weakened. Conclusion A simple and stable rabbit model of AAA can be successfully established by using domestic porcine elastase combined with calcium chloride immersion method.
引文
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[8]Busch A,Chernogubova E,Jin H,et al.Four surgical modifications to the classic elastase perfusion aneurysm model enable haemodynamic alterations and extended elastase perfusion[J].Eur J Vasc Endovasc Surg,2018,56:102-109.
[9]Fahed R,Darsaut TE,Salazkin I,et al.Testing stenting and flow diversion using a surgical elastase-induced complex fusiform aneurysm model[J].AJNR Am J Neuroradiol,2017,38:317-322.
[10]Bi Y,Yu Z,Xu K,et al.Transauricular intra-arterial and intravenous digital subtraction angiography for abdominal aortic aneurysm imaging in a rabbit model[J].Ann Med Surg(Lond),2017,18:24-27.
[11]Freestone T,Turner RJ,Higman DJ,et al.Influence of hypercholesterolemia and adventitial inflammation on the development of aortic aneurysm in rabbits[J].Arterioscler Thromb Vasc Biol,1997,17:10-17.
[12]Tanaka A,Hasegawa T,Chen Z,et al.A novel rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure[J].J Vasc Surg,2009,50:1423-1432.
[13]Verbrugghe P,Verhoeven J,Clijsters M,et al.Creation of abdominal aortic aneurysms in sheep by extrapolation of rodent models:is it feasible?[J].Ann Vasc Surg,2018,52:225-236.
[14]Schulz H,Mohr M,Meyer M.Elastase and papain inhibition by serum of mammals[J].Scand J Clin Lab Invest,1989,49:381-388.
[15]胡国华,符伟国.腹主动脉瘤动物模型的选择[J].中华医学杂志,2015,95:1272-1274.
[2]张明芳,熊吉信,陈锋.腹主动脉瘤发病机制及其动物模型的研究进展[J].中国血管外科杂志·电子版,2010,2:189-192.
[3]严鹏.腹主动脉瘤动物模型研究进展[J].西南国防医药,2017,27:309-311.
[4]Anidjar S,Salzmann JL,Gentric D,et al.Elastase-induced experimental aneurysms in rats[J].Circulation,1990,82:973-981.
[5]汪浩,景在平.腹主动脉瘤动物模型的建立及改进[J].中国现代普通外科进展,2002,5:211-213.
[6]Origuchi N,Shigematsu H,Izumiyama N,et al.Aneurysm induced by periarterial application of elastase heals spontaneously[J].Int Angiol,1998,17:113-119.
[7]Gertz SD,Kurgan A,Eisenberg D.Aneurysm of the rabbit common carotid artery induced by periarterial application of calcium chloride in vivo[J].J Clin Invest,1988,81:649-656.
[8]Busch A,Chernogubova E,Jin H,et al.Four surgical modifications to the classic elastase perfusion aneurysm model enable haemodynamic alterations and extended elastase perfusion[J].Eur J Vasc Endovasc Surg,2018,56:102-109.
[9]Fahed R,Darsaut TE,Salazkin I,et al.Testing stenting and flow diversion using a surgical elastase-induced complex fusiform aneurysm model[J].AJNR Am J Neuroradiol,2017,38:317-322.
[10]Bi Y,Yu Z,Xu K,et al.Transauricular intra-arterial and intravenous digital subtraction angiography for abdominal aortic aneurysm imaging in a rabbit model[J].Ann Med Surg(Lond),2017,18:24-27.
[11]Freestone T,Turner RJ,Higman DJ,et al.Influence of hypercholesterolemia and adventitial inflammation on the development of aortic aneurysm in rabbits[J].Arterioscler Thromb Vasc Biol,1997,17:10-17.
[12]Tanaka A,Hasegawa T,Chen Z,et al.A novel rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure[J].J Vasc Surg,2009,50:1423-1432.
[13]Verbrugghe P,Verhoeven J,Clijsters M,et al.Creation of abdominal aortic aneurysms in sheep by extrapolation of rodent models:is it feasible?[J].Ann Vasc Surg,2018,52:225-236.
[14]Schulz H,Mohr M,Meyer M.Elastase and papain inhibition by serum of mammals[J].Scand J Clin Lab Invest,1989,49:381-388.
[15]胡国华,符伟国.腹主动脉瘤动物模型的选择[J].中华医学杂志,2015,95:1272-1274.