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目的制备携P-选择素单抗的靶向超声微泡(MBp),通过体外流动腔模型评价其在高剪切应力下行血栓超声分子成像的效果。方法采用“亲和素-生物素”桥接法制备MBp和同型对照微泡(MB),应用自制琼脂糖流动腔模型模拟体内高剪切应力血流环境,将MBp和MB随机先后注入琼脂糖模型内,与小鼠血栓孵育30min后,PBS液15cm/s流速不间断冲洗血栓,分别在冲洗2、4、6、8和10min时行对比超声检查并测量声强度(VI)值。免疫组化评价小鼠血栓的P-选择素表达。结果冲洗前MBp组和MB组血栓VI值无明显差异(P>0.05);冲洗后VI值在各时间点MBp组均较MB组大(P<0.05)。冲洗10min后MBp组血栓仍有可视性对比增强,而MB组血栓在冲洗2min后已无可视性对比增强。免疫组化显示血栓表面有明显的P-选择素表达。结论以P-选择素为靶标的MBp在高血流剪切应力下与血栓的靶向结合稳定可靠,可用于动脉血栓的超声分子成像。
Objective To prepare targeted ultrasound microbubbles (MBp) carrying P-selectin monoclonal antibody and to evaluate the effect of ultrasound imaging of thrombus under high shear stress by in vitro flow cavity model. Methods MBp and MB were prepared by “avidin-biotin” bridging method. The home-made agarose flow chamber model was used to simulate the in vivo high shear stress blood flow environment. MBp and MB were injected into agar at random After 30min incubation with thrombus in mice, thrombus was washed with PBS at a flow rate of 15cm / s. Contrast ultrasonography was performed at 2, 4, 6, 8 and 10 min. The intensity of sound (VI) was measured. Immunohistochemical evaluation of P-selectin expression in mouse thrombus. Results There was no significant difference in the VI value of thrombus between MBp group and MB group before washing (P> 0.05). After treatment, the value of VI in MBp group was higher than MB group at each time point (P <0.05). After 10 minutes of flushing, thrombus in MBp group still had more contrast in visibility, while in MB group, there was no enhancement in visibility after 2 minutes of flushing. Immunohistochemistry showed significant thrombosis P-selectin expression. Conclusion P-selectin-targeted MBp has a stable and reliable target binding with thrombus under high shear stress and can be used for ultrasound molecular imaging of arterial thrombosis.