携CCR2靶向超声微泡在乳腺癌模型中的显像研究

Effect of contrast-enhanced ultrosound imaging with CCR2 targeted microbubbles on murine breast tumor model

目的研究携单核细胞趋化因子受体2（CCR2）抗体的靶向微泡在乳腺移植瘤模型中的超声成像效果。方法采用生物素一亲和素桥连法制备CCR2靶向微泡及携同型抗体IgG的对照微泡，并对微泡粒径大小、分布和体外黏附情况：进行研究。同时在乳腺癌皮下移植瘤模型鼠体内进行超声造影实验，比较两种微泡在体内的靶向显影效果，并对肿瘤组织行CCR2免疫荧光检测。结果CCR2靶向微泡形态规整，大小均一性好，分布均匀，与对照微泡相比，具有更高的体外黏附效率，体内靶向黏附信号强度[（6．76±0．26）dB]明显高于对照微泡[（1．06±0．62）dB，P〈0．001]。免疫荧光检测显示肿瘤新生血管内皮细胞见CCR2高表达。结论应用携CCR2抗体的靶向微泡与乳腺癌新生血管内皮靶点特异性结合，有望作为一种新的分子探针评价肿瘤新生血管及监测抗血管治疗的疗效。

Objective To develop and validate a ultrasonographic （US） imaging agent with targeted microbubbles that attaches to chemokine receptor 2 （CCR2） and to compare the US single obtained from targeted microbubble with that from control microbubble in routine breast tumor model. Methods The microbubble which carried CCR2 antibody （MBccR2） and isotype-macthed immunoglobulin G-labled control microbubble （MB l） were prepared. The microbubble size and distribution were assessed by AccuSizer780. Binding specificities of targeted microbubble compared with control microbubble were tested with murine microvascular endothelial ceils （bEnd. 3）. Orthotopic breast tumor model was estabished in BALB/c mice with mouse breast cancer 4T1 cell. In vivo imaging signals of contrast material-enhanced ultrasound by use these two different types of microbubble which were injected respectively into each mouse at random order and 30 rain interval. Tumor tissue was stained for CCR2 and CD31. Results Automatic Particle Sizer showed size uniform of two kinds of microbubbles, and narrow distribution of particle size （mean diameter of about 1 - 2 9m） ,which were not significantly different （ P 〉0.05）. Adhension to bEnd. 3 endothelial cells was significantly higher （P 〈0.001） for MBccR2 （mean, 9.50 ±1.51） than that for MBcontrol （mean,0.01 ± 0.01）. Imaging signal in the murine tumor model was significantly higher for MBccR2 [mean, （6.76 ± 0.26）dB] than that for MB romean, （1.06 ±0.62）dB, P 〈0.001）. Immunofluorescence confirmed expression of CCR2 on tumor vasculature. Conclusions The targeted mierobubbles with CCR2 monoclonal antibody had been successfully prepared, which precisely targeted to CCR2 of tumor angiogenesis in the murine breast cancer xenograft tumor models in vivo. These results suggest that the targeted microbubbles as a kind of ultrasound molecular imaging agent with a better specificity can be used for both evaluating tumor neovascularizatlon and monitoring therapeutic effect of anti-angiogenesis.