全组织包埋免疫荧光染色技术与激光散斑血流成像仪技术在小鼠耳跨区皮瓣动物模型研究中的应用  

The application of Whole-Mounting immnunofluorescent staining and laser speckle flow imaging technology in the study of extended mouse-ear flap model

作　　者：林培森[1] 田磊 肖季婷 马启明 方芳[2] 庄跃宏[3] 谢昀[1] Lin Peisen;Tian Lei;Xiao Jiting;Ma Qiming;Fang Fang;Zhuang Yuehong;Xie Yun(Department of Orthopedic Trauma,the First Clinical Medical College of Fujian Medical University,Fuzhou 350005,China;Experimental Center,School of Pharmacy,Fujian Medical University,Fuzhou 350122,China;Department of Human Anatomy and Tissue Embryology,Fujian Medical University,Fuzhou 350122,China)

机构地区：[1]福建医科大学第一临床医学院创伤骨科,福州350005 [2]福建医科大学药学院实验中心,福州350122 [3]福建医科大学人体解剖与组织胚胎学系,福州350122

出　　处：《中华整形外科杂志》2020年第7期802-809,共8页Chinese Journal of Plastic Surgery

基　　金：福建省自然科学资金(2017J01286,2018J01831);福建省科技创新联合资金项目(2017Y9112)。

摘　　要：目的探讨全组织包埋免疫荧光染色技术及激光散斑血流成像技术在小鼠耳部跨区皮瓣研究中的特点和优势。方法共选取25只ICR小鼠,取其中10只,剪断鼠耳中间及外尾侧血管束,建立跨区耳瓣模型,观察建模3 d后耳瓣血供变化情况,同时观察健侧鼠耳的面积、组织层次厚度及血管分布情况。另取5只小鼠,建跨区耳瓣模型,方法同前。于建模后第3天,获取建模侧鼠耳并将其解剖分为前层皮肤、软骨及后层皮肤,取前层皮肤,采用全组织包埋免疫荧光染色技术对耳内的血管、神经及单核巨噬细胞进行染色,观测耳瓣中血管、神经及单核/巨噬细胞的分布及形态。取剩余10只小鼠,在小鼠耳部中间血管体分叉以上水平剪穿鼠耳,建立延迟跨区耳瓣模型,采用激光散斑血流成像仪观测小鼠耳部的血流变化情况,记录术后即刻、1 d、2 d、3 d、4 d时血管的血流灌注值。结果鼠耳面积约为1.3 cm2,厚度为(0.16±0.04)mm,由外尾侧血管束、中间及内头侧3个血管束供血,在剪断鼠耳中间及外尾侧血管束后可形成跨区皮瓣缺血模型。鼠耳前、后层皮肤及软骨的厚度分别为(88±5)μm、(41±3)μm及(29±2)μm;全组织包埋免疫荧光染色清晰地显示在建模后3 d,choke区域呈放射分布的小血管,直径为(50±6)μm,可见小血管之间扩张弯曲的毛细血管,小鼠耳部神经与动脉呈伴行关系,神经节段攀附至动脉表面,而与静脉并无明显伴行和攀附,扩张、弯曲的动脉内有明显数量的单核巨噬细胞成簇分布,而在动脉外侧仅呈游离散在分布。通过激光散斑血流成像仪可观测到在延迟跨区耳瓣模型后,每个耳瓣内有(6±2)条横向走行的血管管径及血流量有明显增大,术后即刻、1 d、2d、3 d、4d时,横向走行血管的平均血流灌注值分别为(92±11)PU、(136±26)PU、(147±27)Pu及(176±27)PU。结论全组织包埋免疫荧光染色及激光散斑血流成像技�Objective To explore the characteristics and advantages of Whole-Mounting immnunofluorescent staining and laser speckle flow imaging technology in the vascular imaging of mouse ear extended flap.Methods In this study,total of 25 ICR mice were included.Ten ICR mice were cut off the middle and lateral angiosome to establish an extended flap model,and 3 days later,the changes in the blood supply of the ear flap were observed.The ear area,tissue layer thickness and blood vessel distribution in the healthy side were observed at the same time.Obtain the mouse ears 3 days after modeling,and dissect them into three layers,i.e,the anterior skin layer,the cartilage layer and the posterior skin layer.The distribution and morphology of blood vessels,nerves and monocytes/macrophages in the anterior skin layer were stained and detected by the whole-mount immunofluorescence staining.Ten mice were adopted and an incision was made through the ear horizontally above the bifurcation of the middle angiosome of the mouse ear to establish a delayed extended flap model.Then the blood flow changes in the mouse ear were observed by laser speckle flow imaging andt he blood perfusion values were recorded immediately,1 d,2 d,3 d and 4 d after the operation,respectively.Results The area of the mouse ear was about 1.3 cm2,the thickness was about(0.16±0.04)mm,and the blood was supplied by three vascular bundles:the lateral caudal vascular bundle,the middle vascular bundle and the medial cephalic vascular bundle.The thickness of the anterior and posterior skin and cartilage of the mouse ears were(88±5)μm,(41±3)μm and(29±2)μm,respectively.The whole-mount immunofluorescence staining results clearly showed that the diameter of small vessels in the choke area was(50±6)μm on the third day after modeling.It could be seen that the nerve and artery in mouse ear were in concomitant relationship and the nerve segment attached to the surface of the artery without obvious accompany or clinging to the vein.There were a large number of monocyte macr

关 键 词：免疫荧光抗体技术 小鼠 外科皮瓣 血流动力学 

分 类 号：R-332[医药卫生] R764.9