抗血管内皮生长因子MR靶向超顺磁性分子探针的构建及体外大肠癌细胞显像的实验研究

目的 探讨携带纳米铁颗粒的抗血管内皮生长因子(VEGF)分子探针的构建方法及其生物理化性状,以及对体外大肠癌细胞的靶向作用.方法 采用化学交联法构建抗体分子探针,应用十二烷基磺酸钠聚丙烯酰胺凝胶电泳及MR扫描仪检测抗体对比剂的生化及磁学特性.将抗体分子探针与人结肠癌SW620细胞在37℃分别孵育30、60、90 min后进行MR扫描,并进行普鲁士蓝染色验证,信号变化数据组间比较采用单因素方差分析和LSD-t检验;以最佳孵育时间为标准,与骨髓间充质干细胞对照,检测抗体对比剂的靶向增强作用.信号变化数据组间比较采用单因素方差分析和析因设计方差分析.结果 携带纳米铁颗粒的抗VEGF分子探针被成功构建并分离,抗体分子探针的弛豫率为0.0426×10~6 mol/s.免疫细胞荧光及普鲁士蓝染色法证实抗体分子探针可以与高表达VEGF的人结肠癌SW620细胞结合.MRI证明抗体分子探针孵育大肠癌细胞30、60、90 min后T_2信号强度分别为392±7、91±8、264±10,与孵育前(679±12)比较差异有统计学意义(F=4735.489,P<0.01),在孵育60 min后T_2WI及T_2~* WI信号强度值降低最明显.抗体分子探针孵育大肠癌细胞对照实验表明,抗体分子探针孵育大肠癌细胞组与干细胞组T_2信号强度分别为82±7和685±43,差异有统计学意义(t=39.167,P<0.05),抗体分子探针孵育大肠癌细胞组T_2WI及T_2~* WI信号强度显著降低.单纯对比剂孵育大肠癌细胞组与干细胞组比较T_2信号强度分别为419±59和400±41,差异无统计学意义(t=-0.718,P>0.05).结论 携带纳米铁颗粒的抗VEGF分子探针已将肿瘤血管生成的评价发展到受体水平,为肿瘤血管生成的诊断与抗血管生成的治疗提供了新的思路.

The preliminary study of molecular imaging of colorectal cancer cells with superparamagnetic iron oxide-based MR targeting probe containing vascular endothelial growth factor in vitro

Objective To develop a superparamagnetic iron oxide (SPIO)-based MR probe containing vascular endothelial growth factor(VEGF) to investigate their biological and chemical properties and targeting effect of colorectal cancer cells in vitro. Methods The anti-VEGF-SPIO probe was fabricated with VEGF antibody and SPIO through chemical method. Its biological and chemical properties and reflexivity were tested with SDS-PAGE and MRL The SW620 cells incubated with anti-VEGF-SPIO probe for 30, 60 and 90 minutes respectively and compared with marrow mesenchymal stem cell at 37℃. The comparison among groups was conducted by using analysis of variance and LSD-t test. The MRI results were confirmed by the Prussian blue staining. The comparison among groups was performed by analysis of variance and factorial experiment. Results SPIO-based MR probe containing VEGF was successfully contributed and isolated. The reflexivity of anti-VEGF-SPIO probe was 0.0426×10~6 mol/s. The immunofluorescence and prussia blue stain proved high expression of VEGF in SW620 cells. Anti-VEGF-SPIO probe and SW620 cellscombined at 37℃ in vitro MRI proved the SW620 cells incubated with anti-VEGF-SPIO probe appeared hypointense on T_2WI and T_2~* WI. MR signal were 392±7,91±8,264±10 for 30, 60 and 90 minutes respectively, which were statistically different from that before incubation 679±12 (F=4735.489, P< 0.01). The intensity decreased most significantly at 60 minutes in vitro. Its MR signal 82±7 were statistically different compared with marrow mesenchymal stem cell 689±43, t=39.167,P<0.05). While SW620 cells incubated without SPIO were not statistically different compared with marrow mesenchymal stem cell, which were 419±59 and 400±41 respectively(t=-0.718,P>0.05). Conclusion Nanoscale iron particles containing the anti-vascular endothelial growth factor molecular probe can evaluate tumor angiogenesis at the receptor level, which provides a new way of the tumor angiogenesis diagnosis and anti-angiogenesis therapy.