探讨CD31内皮细胞抗体靶向荧光成像在肝段标记中的应用价值。

采用免疫荧光法筛选具有快速结合特性的内皮细胞特异性抗体CD31，使用偶联荧光染料Alexa Fluor 647（AF647）定性及定量评价CD31抗体结合特性。采用体外内皮细胞染色分析CD31荧光抗体定位、半衰期及细胞毒性。单细胞悬液模型分析CD31抗体捕获效率。离体肝脏灌注模型分析CD31抗体灌注剂量及捕获效率的关系。荧光显微镜分析灌注肝段与非灌注区差异并定量分析荧光对比度。两组CD31抗体免疫荧光染色半最大效应浓度（EC₅₀）比较采用t检验。

抗小鼠CD31抗体能在10 s与内皮细胞快速特异性结合。定量分析显示，AF647荧光标记CD31抗体后仍具有快速结合特性；AF647荧光标记CD31抗体后孵育10 s和15 min，EC₅₀值分别为（4.23±0.86）、（0.72±0.10）μg/ml，明显高于纯抗体的（1.21±0.24）、（0.32±0.07）μg/ml（t=5.877，5.928；P<0.05）。细胞染色显示CD31荧光抗体定位于细胞膜及细胞质，半衰期为5 h,无细胞毒性；单细胞悬液结合模型显示抗体捕获效率可达80%。体外灌注模型显示CD31荧光抗体可有效被血管内皮细胞捕获，捕获效率随着抗体浓度加大呈轻度下降趋势，灌注200 ng时捕获效率可达（47±8）%，灌注区域的荧光强度可达到非灌注区的（2.4±0.5）倍。

CD31内皮细胞抗体靶向荧光成像具有安全、有效、快速、特异性标记特点，是肝段标记的一种潜在应用方法。

To evaluate the application of endothelial cell-specific CD31 antibody-targeted fluorescence imaging in liver segment labeling.

The endothelial cell-specific antibody CD31 with rapid binding property was selected by immunofluorescence. The binding characteristics of CD31 antibody were qualitatively and quantitatively evaluated with coupling fluorescent dye Alexa Fluor 647 (AF647). The localization, half-life and cytotoxicity of CD31 fluorescent antibody were analyzed by endothelial cell staining in vitro. The capture efficiency of CD31 antibody was analyzed by single cell suspension model. The relationship between the perfusion dose and capture efficiency of CD31 antibody was assessed in liver perfusion model in vitro. The difference between perfused and non-perfused liver segments was analyzed under fluorescence microscope, and the fluorescence contrast was quantitatively analyzed. The concentration for 50% of maximal effect (EC₅₀) of immunofluorescence staining of CD31 antibody were compared by t test between two groups.

Anti-mouse CD31 antibody could rapidly and specifically bind to endothelial cells within 10 s. Quantitative analysis showed that AF647 labeled-CD31 antibody still possessed rapid binding characteristics. After AF647-labeled CD31 antibody was incubated for 10 s and 15 min, the EC₅₀ were (4.23±0.86) and (0.72±0.10) μg/ml respectively, which were significantly higher than (1.21±0.24) and (0.32±0.07) μg/ml of the pure CD31 antibody (t=5.877, 5.928; P<0.05). Cell staining showed that CD31 fluorescent antibody was localized on cell membrane and in cytoplasm with a half-life of 5 h and no cytotoxicity. Single cell suspension binding model revealed that the antibody capture efficiency reached up to 80%. In vitro perfusion model showed that CD31 fluorescent antibody could be effectively captured by vascular endothelial cells, and the capture efficiency was slightly decreased with the increase of antibody concentration. The capture efficiency reached (47±8)% when the perfusion volume was 200 ng, and the fluorescence intensity in the perfused area was (2.4±0.5) times of that in the non-perfused area.

Endothelial cell-specific CD31 antibody-targeted fluorescence imaging is a safe, effective, rapid and specific labeling tool, which is a potential approach for liver segment labeling.