小鼠自体动静脉内瘘端侧吻合模型的建立与评价

Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice

目的建立小鼠自体动静脉内瘘模型,并对造模效果进行评价。方法采用麻醉后颈外静脉-颈总动脉端侧吻合的方式,将10只8周龄雄性C57BL/6小鼠的左侧颈外静脉及颈总动脉分离并进行自体动静脉端侧吻合手术,右侧仅进行颈外静脉暴露而未行缝合(作为对照),从而建立自体动静脉内瘘动物模型。通过多普勒超声、HE和Masson染色以及免疫组织化学染色观察动静脉内瘘流出段静脉与对侧对照静脉的血流动力学、内膜增生和平滑肌细胞增殖相关蛋白表达情况,评估模型构建效果。结果本研究使用10只小鼠,完成造模9只,成功率为90%。手术当天以及术后7 d、14 d的超声检查结果显示,吻合口附近血液流速与管径大小成线性关系,流速越高提示管径越大,峰值速度和管腔外径之间存在正相关性(P=0.0006,R2=0.8317)。术后14 d的HE染色结果显示,自体动静脉内瘘造模后流出段静脉的平均管腔面积显著减少(P<0.0001),内膜面积显著增加(P<0.0001),内膜中膜面积比及内膜细胞密度均显著提升(P<0.0001);Masson染色结果显示,动静脉内瘘术侧的胶原沉积明显增加,平均染色阳性区域占比显著提升(P<0.0001)。免疫组织化学染色结果显示动静脉内瘘术侧胶原蛋白1(Collagen 1)阳性区域占比明显上调(P<0.0001),α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)阳性区域大量增加(P<0.0001)并集中于新生的内膜区域,增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)阳性细胞明显增多(P<0.0001),提示局部细胞增殖水平增加。结论小鼠自体动静脉内瘘造模具有成功率高、稳定性好、实验成本低等优势,该模型为探寻动静脉内瘘内膜增生的生物学机制提供了良好的载体。

Objective To establish an animal model of autologous arteriovenous fistula in mice and evaluate its effect.Methods The left external jugular vein and common carotid artery of 108-week-old male C57BL/6 mice were separated by end-to-side anastomosis of external jugular vein and common carotid artery after anesthesia,and the right jugular vein was exposed without suture as a control,so as to establish an animal model of internal arteriovenous fistula.Doppler ultrasound,HE and Masson staining and immunohistochemical staining were used to observe the hemodynamics,intimal hyperplasia and protein expression of smooth muscle cell proliferation in the outflow vein of the internal arteriovenous fistula and the contralateral control vein,and to evaluate the effect of model construction.Results A total of 10 mice were selected for this study,and 9 mice were successfully modeled,with a success rate of 90%.Ultrasound examinations were performed on the day of surgery,7 and 14 days after surgery,respectively.The results showed that the flow velocity near the anastomosis was linearly correlated with the diameter of the tube.The higher the flow velocity,the larger the diameter of the tube.There was a positive correlation between peak velocity and lumen diameter(P=0.0006,R2=0.8317).After surgery 14 days,HE staining results showed that after autologous arteriovenous fistula molding,the average lumen area of outflow segment vein was significantly decreased(P<0.0001),the intima area was significantly increased(P<0.0001),the intimal area was significantly increased(P<0.0001).On the surgical side of arteriovenous fistula,collagen deposition was significantly increased,and the proportion of Masson-positive regions was significantly increased(P<0.0001).Immunohistochemical staining showed that the proportion of collagen 1 positive areas on the surgical side of arteriovenous fistula was significantly upregulated(P<0.0001),andα-smooth muscle actin(α-SMA),proliferating cell nuclear antigen(PCNA)positive cells increased significantly(P<0.0001),indicating an increase in local cell proliferation level.Conclusion The established mouse autologous arteriovenous fistula model has the advantages of high success rate,good stability and low cost.The model provides a good carrier for exploring the biological mechanism of intimal hyperplasia in arteriovenous fistulas.