CAJ | 학술논문

目的构建一种靶向肝癌磷脂酰肌醇蛋白聚糖-3 (GPC3)的磁共振(MR)分子探针,并探讨其靶向肝癌HepG2细胞的特异性及体外细胞MR成像的可行性.方法用双乳化溶剂挥发法制备聚乳酸-羟基乙酸共聚物(PLGA)纳米粒,在纳米粒表面连接GPC3抗体及顺磁性对比剂Gd3+构建靶向肝癌GPC3的MR分子探针,利用荧光显微镜、电镜、Malvem激光粒径测量仪、电感偶合等离子体原子发射光谱仪及1.5T MR扫描仪观察其表征；利用激光共聚焦显微镜观察该探针与GPC3结合的特异性；利用MR扫描仪观察该探针标记肝癌HepG2细胞后的体外MR成像能力.多组均数间用方差分析进行比较,组内两均数间用LSD-t检验进行比较.结果成功构建了靶向肝癌GPC3的MR分子探针GPC3抗体-PLGA-Gd纳米粒,其形态规则、呈球形,粒径(495.0±17.5)nm,大小、分布均匀,分散性好,无明显聚集.经电感偶合等离子体原子发射光谱仪测定,1 molPLGA上大约载有12 mol的Gd3+.随着Gd3+浓度的增加,MR扫描时的SNR值相应增加,组间SNR值差异具有统计学意义(F=1721.131,P＜0.05)；体外HepG2细胞寻靶后行MR成像并计算其相应SNR,靶向组SNR值为3.45±0.21,非靶向组SNR值为1.43±0.07,对照组SNR值为1.12±0.03,靶向组的SNR值明显高于非靶向组及对照组(LSD-t检验,P值均＜0.05),非靶向组与对照组的SNR值差异无统计学意义(P＞0.05).结论应用PLGA纳米粒、GPC3抗体及顺磁性对比剂Gd3+构建的靶向肝癌GPC3的MR分子探针在体外能与HepG2细胞特异性结合,且标记HepG2细胞后能在1.5T MR扫描仪上成像,有望应用于活体肝癌的特异性成像,为肝癌的早期诊断提供一种无创的成像手段.
목적 구건일충파향간암린지선기순단백취당-3 (GPC3)적자공진(MR)분자탐침,병탐토기파향간암HepG2세포적특이성급체외세포MR성상적가행성.방법 용쌍유화용제휘발법제비취유산-간기을산공취물(PLGA)납미립,재납미립표면련접GPC3항체급순자성대비제Gd3+구건파향간암GPC3적MR분자탐침,이용형광현미경、전경、Malvem격광립경측량의、전감우합등리자체원자발사광보의급1.5T MR소묘의관찰기표정；이용격광공취초현미경관찰해탐침여GPC3결합적특이성；이용MR소묘의관찰해탐침표기간암HepG2세포후적체외MR성상능력.다조균수간용방차분석진행비교,조내량균수간용LSD-t검험진행비교.결과 성공구건료파향간암GPC3적MR분자탐침GPC3항체-PLGA-Gd납미립,기형태규칙、정구형,립경(495.0±17.5)nm,대소、분포균균,분산성호,무명현취집.경전감우합등리자체원자발사광보의측정,1 molPLGA상대약재유12 mol적Gd3+.수착Gd3+농도적증가,MR소묘시적SNR치상응증가,조간SNR치차이구유통계학의의(F=1721.131,P＜0.05)；체외HepG2세포심파후행MR성상병계산기상응SNR,파향조SNR치위3.45±0.21,비파향조SNR치위1.43±0.07,대조조SNR치위1.12±0.03,파향조적SNR치명현고우비파향조급대조조(LSD-t검험,P치균＜0.05),비파향조여대조조적SNR치차이무통계학의의(P＞0.05).결론 응용PLGA납미립、GPC3항체급순자성대비제Gd3+구건적파향간암GPC3적MR분자탐침재체외능여HepG2세포특이성결합,차표기HepG2세포후능재1.5T MR소묘의상성상,유망응용우활체간암적특이성성상,위간암적조기진단제공일충무창적성상수단.
Objective To prepare a glypican-3 (GPC3)-targeting hepatocellular carcinoma MR molecular probe and to evaluate its targeting specificity using HepG2 cells.Methods Poly(lactic-coglycolic acid) (PLGA) nanoparticles were prepared by a double emulsion solvent evaporation method,and the surfaces were connected with anti-GPC3 mono-antibody and paramagnetic substance Gd3+.The physical properties of the probes were investigated using fluorescence microscopy,electron microscopy,Malvern particle size analysis,inductively coupled plasma atomic emission spectroscopy (ICP-AES) and 1.5T MR imaging.The specificity of the probes to target cultured HepG2 cells was determined by laser confocal microscopy.The signal characteristics,including signal-to-noise ratio (SNR),after co-incubation with HepG2 cells were analyzed by 1.5T MR imaging.Significance of differences between multiple groups (target group,non-target group,and control group) was assessed by one-way analysis of variance,and between two groups was assessed by the LSD-t test.A difference was considered to be statistically significant at P ＜ 0.05.Results The GPC3-targeting hepatocellular carcinoma MR molecular probes were successfully prepared.The nanoparticles had a spherical shape,size of 495 ± 17.5 nm,uniform size distribution,good dispersibility,no obvious aggregation,and could significantly increase the T1 signal.Using the ICP-AES measurement,1 mol PLGA carried about 12 mol Gd3+,and as the Gd3+ concentration increased,the T1 signal increased.The prepared MR molecular probes could specifically target HepG2 cells,and could increase the T1 signal.The SNR value of the target group was 3.45 ± 0.21,of the non-target group was 1.43 ± 0.07,and of the control group was 1.12 ± 0.03.The SNR value of the target group was significantly higher than that of the non-target group and the control group (P ＜ 0.05); there was no significant difference between the non-target group and the control group (P ＞ 0.05).Conclusion PLGA nanoparticles,anti-GPC3 mono-antibody and paramagnetic Gd3+ can be used to successfully prepare GPC3-targeting hepatocellular carcinoma MR molecular probes which are capable of specifically targeting HepG2 cells in vitro and being detected by 1.5T MR imaging.These MR molecular probes may represent a useful noninvasive imaging method for detecting early hepatocellular carcinoma in vivo.