骨肉瘤血管内皮细胞特异性结合短肽的体内功能验证  

The functional identification of short peptide binding specifically to osteosarcoma-associated vascular endothelial cells in-vivo

作　　者：杨澜波[1] 史占军[1] 管明强[1] 李朋[1] 赵赞栋[1] 王健[1] 肖军[1] 

机构地区：[1]南方医科大学南方医院脊柱骨病外科,广州510515

出　　处：《中华骨科杂志》2010年第5期501-505,共5页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金（30371450）;广东省自然科学基金（32948）;广东省科技计划（20088030301348）

摘　　要：目的验证骨肉瘤血管内皮细胞（osteosarcoma—associated vascular endothelial cells，OAVECs）特异性结合肽（TKPDKGY）对荷瘤裸鼠模型体内OAVECs的亲和性。方法合成短肽（TKPD—KGY）并在其N端加载一个异硫氰酸荧光素（fluorescein isothiocyanate，FITC），纯化后将短肽荧光复合物注入荷瘤裸鼠血管内，通过小动物整体荧光成像来观察短肽在活体动物内的分布，同时定时处死裸鼠，切取重要器官和肿瘤制冰冻切片，使用激光共聚焦显微镜扫描来评价短肽在体内与OAVECs的亲和性。结果小动物整体荧光成像显示荷瘤裸鼠注射FITC-TKPDKGY 10min后，肿瘤处即可见微弱荧光富集，30min时显示明显并逐渐增强，1h后达最强，4h后逐渐消退，至24h基本无荧光残留。激光共聚焦扫描可见肿瘤血管和血管丛荧光强度较强，肿瘤组织仅见少量短肽结合，可提示有一定的结合性。心、脑、肺和肝组织显示并不明显。肾脏镜下可见肾血管并无荧光显像，但肾小球和肾小管部可见荧光富集，提示短肽在此代谢。结论TKPDKGY对目标细胞有良好的靶向性，为短肽进一步结合效应分子应用于骨肉瘤的诊断和治疗奠定一定的实验基础。Objective To identify the binging specification of the short peptide （TKPDKGY） specifically binding to osteosarcoma-associated vascular endothelial cells（OAVECs） by using BALB/c-nu mice with osteosarcoma. Methods The TKPDKGY was synthesized with the N-terminal marked with fluorescein isoth- iocyanate（FITC）. After purification, the peptide was injected into the vessel of nude mice. Whole-body optical imaging system was used to investigate the peptide distribution in mice. Four hours after injection, the nude mice were sacrificed. The vital organs and tumor of mice were used to make frozen slice. Confocal microscope was used to detect the in-vivo binding activity of the short peptide. Results Whole-body optical imaging system showed that fluorescence at tumor site was weak at 10 min after injection, and gradually increased at 30 rain, reached the peak at 1 h, then subsidised 4 h later, and no residual fluorescence at 24 h. Confocal microscope also to shown that the short peptide homed to vascular endothelium in osteosarcoma but was not detectable in the heart, brain, lung and liver. The fluorescence at glomerular and tubular was detected by confocal microscope but not renal vascular endothelium which means that the kidney is metabolism organ of TKPDKGY. Conclusion The TKPDKGY peptide has the specifically binding activity to vascular endothelium in osteosarcoma. It may be used as good targeting vector for selective delivery of therapeutics and as a diagnostic probe for the detection of osteosarcoma.

关 键 词：骨肉瘤 内皮细胞 C肽 

分 类 号：R738.1[医药卫生—肿瘤]