CAJ | 학술논문

目的:探讨制备靶向结合活化血小板的脂质超声造影剂的新方法,评价制备的靶向超声造影剂体外与血栓靶向结合的能力.方法:首先合成荧光标记的,能与血小板膜糖蛋白GPⅡb/Ⅲa受体特异性结合的赖氨酸-甘氨酸-天冬氨酸-丝氨酸多肽-棕榈酸化合物(KGDS-Palm).采用"超声-高速剪切"法,以带荧光FITC的KGDS-Palm为主要原料之一,制备靶向脂质超声造影剂.马尔文公司粒径测定仪检测微泡大小及分布;Coulter计数仪分析浓度;荧光显微镜下观察KGDS多肽与微泡的结合情况;流式细胞仪评价多肽与微泡的结合效率;观察靶向微泡在体外的稳定性;采用体外血栓模型,检测靶向微泡与血栓靶向结合的特异性.结果:KGDS靶向超声造影剂呈淡黄色混悬液,微泡浓度约为1.5×10~9/mL,平均粒径为1.5 μm,98%的微泡小于5 μm.荧光显微镜显示靶向超声造影剂表面呈明亮的绿色荧光;流式细胞仪检测KGDS结合效率为90.04%;体外4 ℃保存48 h后微泡浓度及粒径无显著改变;体外靶向及其拮抗实验证实,靶向超声造影剂与血栓特异性结合.结论:采用"超声-高速剪切法"制备靶向超声造影剂,方法简单易行,有利于靶向造影剂的制备及纯化;KGDS靶向超声造影剂稳定性好、靶向结合特异性强.
목적:탐토제비파향결합활화혈소판적지질초성조영제적신방법,평개제비적파향초성조영제체외여혈전파향결합적능력.방법:수선합성형광표기적,능여혈소판막당단백GPⅡb/Ⅲa수체특이성결합적뢰안산-감안산-천동안산-사안산다태-종려산화합물(KGDS-Palm).채용"초성-고속전절"법,이대형광FITC적KGDS-Palm위주요원료지일,제비파향지질초성조영제.마이문공사립경측정의검측미포대소급분포;Coulter계수의분석농도;형광현미경하관찰KGDS다태여미포적결합정황;류식세포의평개다태여미포적결합효솔;관찰파향미포재체외적은정성;채용체외혈전모형,검측파향미포여혈전파향결합적특이성.결과:KGDS파향초성조영제정담황색혼현액,미포농도약위1.5×10~9/mL,평균립경위1.5 μm,98%적미포소우5 μm.형광현미경현시파향초성조영제표면정명량적록색형광;류식세포의검측KGDS결합효솔위90.04%;체외4 ℃보존48 h후미포농도급립경무현저개변;체외파향급기길항실험증실,파향초성조영제여혈전특이성결합.결론:채용"초성-고속전절법"제비파향초성조영제,방법간단역행,유리우파향조영제적제비급순화;KGDS파향초성조영제은정성호、파향결합특이성강.
Objective To prepare a thrombus-targeted ultrasonic contrast agent and to investigate its targeted ability to fresh blood clots. Methods We first synthesized FITC-KGDS-Palm compound, and then prepared thrombus-targeted microbubbles using "ultrasound & high speed shearing method".Fluorescence labeling thrombus-specific peptides and KGDS,directed at the activated glycoprotein(GP)Ⅱb/Ⅲa receptor of platelets were attached to the surface of lipid microbubbles. The concentration and size of TUCA were measured by Malvern Zeta Sizer Nano-ZS590 and Coulter counter.Immunofluorescence was applied to confirm the conjugation.The conjunct ratio was assessed by flow cytometer (FCM).Results The KGDS-TUCA was straw yellow turbid liquor,and the concentration was 1.5×10~9/mL,and the average size was 1.5 μm. The targeted microbubbles conjugated with the thrombus-specific peptides showed bright green rings by fluorescence microscope.FCM demonstrated that the wavelength of shell of KGDS-TUCA changed greatly,and the conjunct ratio was 90.04%.In vitro study showed KGDS-TUCA remained stable for 48 h at 4 ℃ and target-attached to blood clots and showed good stability.Conclusion The ultrasound & high speed shearing method to prepare TUCA is easy and in favor of purification.KGDS-TUCA has high specific biological activity.The conjunct ratio and stability of KGDS-TUCA are excellent.