以短肽K237为配体的靶向脂质体超声造影剂构建方法  

Preparation of liposome ultrasonic contrast agent with ligand peptide K237

作　　者：何洁[1] 杨莉[2] 李颖嘉[1] 孙学刚[3] 刁建新[3] 李传刚[4] 宾建平[4] 龚渭冰[1] 

机构地区：[1]南方医科大学南方医院超声科,广东广州510515 [2]南方医科大学药学部,广东广州510515 [3]南方医科大学中医学院,广东广州510515 [4]南方医科大学南方医院心内科,广东广州510515

出　　处：《中国医学影像技术》2011年第1期3-7,共5页Chinese Journal of Medical Imaging Technology

基　　金：国家自然科学基金资助面上项目(30670580);广东省博士启动基金(9451051501003761)

摘　　要：目的探讨以与血管内皮生长因子(VEGF)的主要受体KDR特异性结合的短肽K237(P)为配体制备靶向脂质体超声造影剂(P-Bio-Av-Bio-Mbs)的方法。方法 采用生物素-亲和素桥接法构建P-Bio-Av-Bio-Mbs,流式细胞术筛选最佳配体适配剂量,光镜及荧光显微镜观察靶向微泡与KDR强阳性表达的人大肠癌LOVO细胞结合情况,计算花环形成率。分别以5、50、99ml/h速率水流冲刷,光镜观察靶向微泡与LOVO细胞结合情况。结果 不同亲和素剂量下(0、2、6、10、30μg),微泡表面亲和素携带率差异有统计学意义(P<0.05)。Mavidin=6μg时,携带率增长达平台期;不同短肽剂量下(0、30、40、50、60、70、100μg),微泡表面短肽携带率差异有统计学意义(P<0.05),当MK237=50μg时,微泡表面短肽携带率增长达平台期。光镜下KDR强阳性表达的LOVO细胞周围花环形成率高达90.52%,荧光显微镜下微泡外壳发出明亮绿色荧光。随冲刷速度增加,靶细胞周围黏附的靶向微泡减少,在99ml/h冲刷速度下,靶细胞周围仍可见花环结构。结论 通过生物素-亲和素桥连作用,短肽K237被有效装配在P-Bio-Av-Bio-Mbs表面,体外具有靶向特异性及一定稳定性。流式细胞术是筛选靶向微泡配体适配剂量的可靠方法。Objective To assess preparation method of a new kind of targeted liposome ultrasonic contrast agent with small peptide K237 as the ligand which can combine specifically with KDR as the main receptor of VEGF.Methods Targeted bubbles（P-Bio-Av-Bio-Mbs） were formed through ＂biotin-avidin＂ bridge grafting.Flow cytometry screening was performed to explore the best dose of the ligands,then targeted-bubbles were incubated respectively with LOVO and LS174T which were KDR expressed in different cells.Meanwhile,rosette formation rate was calculated.Results The bubble surface＇s avidin-carrying rates were significant different（P0.05） on different dosages of avidin（0,2,6,10,30 μg）.When M_avidin=6 μg,the avidin labelling ratio reached plateau.There were significant differences in the ratio of peptide K237 labelling（P0.05） as different peptide dosages（0,30,40,50,60,70,100 μg）.When M_k237=50 μg,the peptide labelling ratio reached plateau.In KDR sharply positive expressed LOVO cells,the surrounding rosette formation rate was as high as 90.52% with strong fluorescence intensity.Targeted microbubbles decreased as the water velocity increased.When the velocity reached 99 ml/h,rosette formation still could be seen surrounding the targeted cells.Conclusion KDR-targeted liposome contrast agent with small peptide liganded has been successfully prepared through biotin-avidin mediation,and showed special targeting ability and stability in vitro.Flow cytometry can quantitatively analyze the best dose of ligands carrying targeted microbubbles.

关 键 词：微泡 流式细胞术 

分 类 号：Q784[生物学—分子生物学]