目的：应用剪碎和混合酶消化法，建立一种有效的用于流式细胞术分析的小鼠血管组织单细胞悬液，用于检测血管紧张素Ⅱ刺激引起的血管组织中炎性细胞的浸润。方法采用皮下微量泵持续泵入血管紧张素Ⅱ(1000 ng·kg-1·min-1)，于灌注3 d后将小鼠麻醉后立即处死，取出胸主动脉和腹主动脉，迅速剪碎，用混合酶消化小鼠血管组织，制备单细胞悬液，用于流式细胞技术检测。结果每只小鼠血管组织分离细胞数可达5×105个/mL；光镜下单细胞悬液细胞完整透明，分散排列，状态良好；流式细胞术检测显示血管紧张素Ⅱ处理后明显增加了血管组织中中性粒细胞、T淋巴细胞和巨噬细胞的数量。结论应用剪碎和混合酶联合消化法制备小鼠血管组织单细胞悬液简便可行，为血管炎性细胞浸润的研究提供了有效的组织单细胞悬液制备方法。%Objective To set up an effective method using flow cytometry technology to detect the infiltration of inflammatory cells in murine aorta by preparation of single cell suspensions. Methods Osmotic mini pumps with angiotensin Ⅱ(1 000 ng·kg-1 ·min-1 ) were implanted subcutaneously into mice for 3 d. The mice were sacrificed immediately after anesthesia, and the thoracic and abdominal aorta was cut off immediately after it was taken out. The vascular tissue of mice was digested with the digestive enzyme to prepare single cell suspension for flow cytometry analysis. Results 5×105 cells/mL could be isolated from each vascular tissue of mouse, and more intact cells and better viability were found in the single cell suspension; The results of flow cytometry showed angiotension Ⅱ treatment increased the number of neutrophisl, T lymphocyte cells and macrophages in the aorta. Conclusion This method is a simple, effective, stable and reliable one for the preparation of single-cell suspension of murine aorta, which will help to study the infiltration of inflammatory cells in murine aorta.
展开▼
