L5多肽介导的链霉亲和素－聚乙二醇－超小超顺磁性氧化铁纳米复合物对磷脂酰肌醇蛋白聚糖－3阳性肝细胞癌细胞进行预定位MRI的实验研究  

In vitro studies of glypiean-3 targeting pretargeting technology for molecular MRI of hepatocellular carcinoma

作　　者：李维粤[1] 许乙凯[1] 洪少馥[2] 陈婉琪[1] 马立超[1] 严承功[1] 吴元魁[1] 

机构地区：[1]南方医科大学南方医院影像中心,广州510515 [2]南方医科大学南方医院超声科,广州510515

出　　处：《中华放射学杂志》2015年第12期935-940,共6页Chinese Journal of Radiology

基　　金：广东省自然科学基金（S2013010015689）

摘　　要：目的探讨生物素化L5（L5-BT）多肽介导的链霉亲和素－聚乙二醇－超小超顺磁性氧化铁（SA－PEG－USPIO）纳米复合物对磷脂酰肌醇蛋白聚糖-3（GPC3）阳性肝癌细胞进行预定位体外MRI的价值。方法用羧基荧光素（FAM）修饰的L5多肽（L5-FAM）对HepG2肝癌细胞及HL-7702正常肝细胞进行免疫荧光染色，并进行竞争抑制实验，在倒置荧光显微镜下观察荧光表达。采用间接免疫荧光法验证GPC3表达。制备SA-PEG-USPIO纳米复合物，并以聚乙二醇-超小超顺磁性氧化铁（PEG-USPIO）为对照测定两者的水合粒径、表面电位及T2弛豫率，并体外测定二者的细胞毒性。将HepG2细胞和HL-7702细胞按照加人试剂的不同分为L5-BT介导SA-PEG-USPIO组、SA—PEG—USPIO组和空白组共3组，行普鲁士蓝染色，并行体外MRI，记录标准化T2信号强度。采用单因素方差分析比较3组间的标准化B信号强度差异。结果倒置荧光显微镜下，多肽L5出现在HepG2细胞表面及胞质内，竞争实验组及HL-7702细胞组荧光明显减少，HepG2肝癌细胞有较强的GPC3阳性表达染色。分子探针SA-PEG-USPIO构建成功。SA-PEG-USPIO水合粒径为（35．97±5．19）nm，分散系数（PdZ）为（0．17±0．05），Zeta电位为（-7．91±1．22）mV；PEG-USPIO水合粒径为（22．73±3．31）nm，PdI为（0．21±0．02），Zeta电位为（4．22±0．53）mV。SA-PEG-USPIO和PEG—USPIO的T2弛豫率分别为0．1039×10^3和0．1394×10^3mM^-1s^-1。Fe浓度达到2．4mmol／L时，HL7702细胞存活率均可达到80％以上。普鲁士蓝染色显示，L5-BT介导SA-PEG-USPIO组中HepG2细胞的胞膜、胞质内可见大量蓝染铁颗粒，其他各组明显减少。体外MRI，L5-BT介导SA-PEG-USPIO组、SA—PEG-USPIO组和空白组的HepG2细胞标准化T2WI信号强度分别为39±7、77±12、93±4，差异有统计学意义（P〈0．01）；各组HL-7702细胞的标准化T2WI信号强度分别为69±11、78±8�Objective To explore the value of pretargeting technology in vitro MRI of L5 peptide guided streptavidin-conjugated and polyethylene glycol modification protected ultra-small superparamagnetic iron oxide（SA-PEG-USPIO） to hepatocellular carcinoma（HCC） via glypican-3（GPC3） receptor. Methods Direct immumofluorescence assay with carboxyfluorescein（FAM） labeled L5 and competitive inhibition was performed in HepG2 and HL-7702 cells. Imaging was obtained from fluorescent microscope. Immunoassay fluorescence images were carried out to determine the expression of GPC3 in HepG2 cell. PEG-USPIO conjugated with streptavidin was made by carbodiimide reaction, and the hydrodynamic diameters, Zeta potential and magnetic relaxivity of SA-PEG-USPIO and PEG-USPIO were measured. HL7702 cells were used for evaluate cells viability of SA-PEG-USPIO and PEG-USPIO. HepG2 and HL-7702 cells were used as experimental and control group respectively. Each of the two cell lines were further divided into three groups： L5-BT united SA-PEG-USPIO group, SA-PEG-USPIO group and control group. Prussian blue staining and MRI was preformed to observe the targeting efficacy of SA-PEG-USPIO respectively, and normalized T2 signal was recorded. The significant changes of normalized T2 signal intensity among groups was determine by using One-way analysis of varianee. Results There were much more fluorescences on the membrane and cytoplasm of HepG2 cells than those on HL-7702 cells and cells of competition group. And indirect immunofluorescence images show the obvious expression of GPC3 in HepG2 cell. The SA-PEG-USPIO and PEG-USPIO nanoparticles had hydrodynamic diameters of （22.73 ± 3.31） and （35.97 ± 5.19）nm, Zeta potential of them were （4.22 ±0.53） and （- 7.91 ±1.22）mV and magnetic relaxivity were 0.139 4± 103 and 0.103 9± 10^3 mM^-1s^-1. Although the highest concentration of SA-PEG-USPIO and PEG-USPIO was 2.4 mmol/L, cells viability was greater than 80%. The most iron particle was observed in L5-BT united SA-PEG-U

关 键 词：癌 肝细胞 磁共振成像 分子探针 

分 类 号：R735.7[医药卫生—肿瘤]