小鼠自体动静脉内瘘端侧吻合模型的建立与评价

doi:10.12300/j.issn.1674-5817.2023.093

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (6): 595-603.DOI: 10.12300/j.issn.1674-5817.2023.093

小鼠自体动静脉内瘘端侧吻合模型的建立与评价

刘欣¹^,²^,³, 石少波¹^,²^,³, 张翠¹^,²^,³, 杨波¹^,²^,³, 曲川¹^,²^,³()()

^1.武汉大学心血管病研究所, 武汉 430060
^2.心血管病湖北省重点实验室, 武汉 430060
^3.武汉大学人民医院心内科, 武汉 430060

收稿日期:2023-06-30 修回日期:2023-10-18 出版日期:2023-12-25 发布日期:2024-01-06
通讯作者: 曲川（1988—），男，博士，主治医师，研究方向：血管增生的基础研究。E-mail： 175650787@qq.com。ORCID： 0009-0002-0430-7416
作者简介:刘欣（1992—），女，博士，主治医师，主要从事大血管疾病的基础研究。E-mail: RM003805@whu.edu.cn
基金资助:
国家自然科学基金资助项目“IL-7/IL-7R调控巨噬细胞炎症反应在腹主动脉瘤发病中的作用及机制研究”(82200520);武汉市知识创新专项-曙光计划资助项目“IL-7/IL7R对腹主动脉瘤中巨噬细胞迁移、浸润和极化对作用及机制研究”(202202080102);中央高校基本科研业务费专项资金资助项目“Grem1调控巨噬细胞凋亡在小鼠腹主动脉瘤中的作用及机制”(2042022kf1085)

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

Xin LIU¹^,²^,³, Shaobo SHI¹^,²^,³, Cui ZHANG¹^,²^,³, Bo YANG¹^,²^,³, Chuan QU¹^,²^,³()()

^1.Cardiovascular Research Institute, Wuhan University, Wuhan 430060, China
^2.Hubei Key Laboratory of Cardiology, Wuhan 430060, China
^3.Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, China

Received:2023-06-30 Revised:2023-10-18 Published:2023-12-25 Online:2024-01-06
Contact: QU Chuan (ORCID: 0009-0002-0430-7416), E-mail: 175650787@qq.com

摘要/Abstract

摘要：

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=0.000 6，R²=0.831 7）。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＜0.000 1），内膜面积显著增加（P＜0.000 1），内膜中膜面积比及内膜细胞密度均显著提升（P＜0.000 1）；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＜0.000 1）。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＜0.000 1），α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）阳性区域大量增加（P＜0.000 1）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＜0.000 1），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。

关键词: 动静脉内瘘, 动物模型, 尿毒症, 血液透析, 内膜增生, 小鼠

Abstract:

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 10 8-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=0.000 6, R²=0.831 7). 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 < 0.000 1), the intima area was significantly increased (P < 0.000 1), the intimal area was significantly increased (P < 0.000 1). On the surgical side of arteriovenous fistula, collagen deposition was significantly increased, and the proportion of Masson-positive regions was significantly increased (P < 0.000 1). Immunohistochemical staining showed that the proportion of collagen 1 positive areas on the surgical side of arteriovenous fistula was significantly upregulated (P < 0.000 1), and α-smooth muscle actin (α-SMA) , proliferating cell nuclear antigen (PCNA) positive cells increased significantly (P < 0.000 1), 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.

Key words: Arteriovenous fistula, Animal model, Uremia, Hemodialysis, Intimal hyperplasia, Mice

中图分类号:

Q95-33

刘欣, 石少波, 张翠, 杨波, 曲川. 小鼠自体动静脉内瘘端侧吻合模型的建立与评价[J]. 实验动物与比较医学, 2023, 43(6): 595-603.

Xin LIU, Shaobo SHI, Cui ZHANG, Bo YANG, Chuan QU. Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 595-603.

图/表 6

图1 小鼠自体动静脉内瘘模型手术示意图注：红色血管代表颈总动脉，蓝色血管代表颈外静脉。A，分离颈外静脉及颈总动脉并制作切口；B，动静脉端侧吻合。

Figure 1 Operation diagram of autologous arteriovenous fistula in miceNote：The red blood vessel represents the common carotid artery and the blue blood vessel represents the external jugular vein. A， The external jugular vein and common carotid artery were separated， and incisions were made； B， Arteriovenous end-to-side anastomosis.

图2 小鼠颈外静脉与颈总动脉端侧吻合步骤注：A，分离颈外静脉与颈总动脉，结扎颈外静脉远心端，使用显微血管夹夹闭颈外静脉近心端和颈总动脉近远心端；B，缝合吻合口0点与12点方向后，连续缝合吻合口前壁；C，提拉翻转静脉，连续缝合吻合口后壁；D，开放血管夹后可见静脉端充盈，检查渗血情况。

Figure 2 Procedures of end-to-side anastomosis of external jugular vein and common carotid artery in miceNote：A， The external jugular vein and the common carotid artery were separated， the distal end of the external jugular vein was ligated， and the proximal end of the external jugular vein as well as the proximal and distal end of the common carotid artery were closed with a microvascular clamp； B， Sutured the anterior wall of the anastomosis continuously at 0 and 12 points of the anastomosis； C， Lifted the inverted vein and sutured the posterior wall of the anastomosis continuously； D， After opening the vascular clamp， it could be observed that the end of the vein was full. Check for any blood seepage.

图3 小鼠自体动静脉内瘘模型的无创多普勒超声评估注：A，术后14 d的吻合口超声流速代表图；B，术后当天以及术后7 d、14 d小鼠吻合口流速监测数据（n=9）；C，术后14 d小鼠吻合口流速与动静脉内瘘流出道外径的关系（n=9）。

Figure 3 Noninvasive Doppler ultrasound assessment in mice autologous arteriovenous fistula （AVF） modelNote：A, Anastomotic ultrasound flow rate after surgery 14 days; B, The anastomotic flow rate was monitored on the day after surgery and 7 days and 14 days after surgery (n=9); C, The relationship between anastomotic flow velocity and outflow diameter of arteriovenous fistula after surgery 14 days (n=9).

图4 小鼠自体动静脉内瘘（AVF）模型的血管内膜分析注：A，术后14 d对照侧和AVF侧流出静脉的HE切片代表性图片，比例尺= 250 μm；B，术后14 d AVF侧管腔面积相比于对照侧明显缩小（n=5）；C，术后14 d AVF侧内膜面积相比于对照侧显著增大（n=5）；D，术后14 d AVF侧内膜中膜比相比于对照侧显著升高（n=5）；E，术后14 d AVF侧内膜细胞密度相比于对照侧显著增加（n=5）。

Figure 4 Analysis of vascular intima in mice autologous arteriovenous fistula （AVF） modelNote：A， the representative pictures of HE sections of the outflow veins on the control side and the AVF side at 14 days after surgery， the scale = 250 μm； B， the lumen area on the AVF side was significantly smaller than that on the control side at 14 days after surgery （n=5）； C， the intimal area on the AVF side was significantly larger than that on the control side at 14 days after surgery （n=5）； D， the intima-media ratio on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）； E， the intimal cell density on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）.

图5 小鼠自体动静脉内瘘模型的血管胶原沉积分析注：A，术后14 d对照侧和AVF侧流出静脉的Masson染色以及胶原蛋白1（Collagen 1）免疫组织化学检测代表性图片，比例尺=250 μm；B，术后14 d AVF侧Masson染色阳性区域比例相比于对照侧明显上调（n=5）；C，术后14 d AVF侧Collagen 1阳性区域比例相比于对照侧明显升高（n=5）。

Figure 5 Analysis of collagen deposition in mice autologous arteriovenous fistula （AVF） modelNote： A. Masson staining and representative images of Collagen 1 immunohistochemical detection of outflow veins on the control side and AVF side at 14 days after surgery （scale = 250 μm）； B， the proportion of MASSON-positive areas on AVF side at 14 days after surgery was significantly higher than that on the control side （n=5）； C， the proportion of Collagen 1 positive areas on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）.

图6 小鼠自体动静脉内瘘模型中α-SMA和PCNA表达分析注：A，术后14 d对照侧和AVF侧流出静脉的α-平滑肌肌动蛋白（α-SMA）和增殖细胞核抗原（PCNA）免疫组织化学检测代表性图片，比例尺=250 μm；B，术后14 d AVF侧α-SMA阳性细胞比例明显上调（n=5）；C，术后14 d AVF侧PCNA阳性细胞比例明显升高（n=5）。

Figure 6 α-SMA and PCNA analysis in mice autologous arteriovenous fistula （AVF） modelNote： The expression of α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) in the outflow vein on the control side and AVF side at 14 days after surgery was detected by immunohistochemistry, scale =250 μm; B, the proportion of α-SMA positive cells on AVF side was significantly up-regulated at 14 days after surgery (n=5); C, the proportion of PCNA positive cells on AVF side increased significantly at 14 days after surgery (n=5).

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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

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