摘要目的 通过制备131I-NGR进行人CD13表达细胞株的体外筛选,探讨NGR对不同肿瘤的靶向性.方法 设计合成NGR短肽,以Iodogen法对NGR进行131I标记,探讨Iodogen法标记的最佳实验条件及标记物性质.将20μl标记产物置于双倍体积的生理盐水和新鲜人血清中混合,在室温和37℃下分别于2、12和24 h时用TLC测定放化纯,评价产物的体外稳定性.通过细胞免疫荧光实验和蛋白印迹实验检测HT-1080、HUVEC、HepG2、U87Mg、PC-3、HT-29和MCF-7细胞株的CD13表达.对阳性表达细胞株进行131I-NGR受体分析.以四甲基偶氮唑蓝(MTT)法测定不同浓度Na131I、NGR和131I-NGR在24、48和72 h对HT-1080和HT-29细胞株的体外生长抑制率,采用单因素方差分析进行统计分析.结果 成功标记131I-NGR,最佳标记条件为131I 18.5 MBq、NGR 10μg和Iodogen 20 μg,标记率为(93.7±2.5)％,比活度达1.41 TBq/mmol.标记后稳定性良好,24 h后标记率仍＞87％.免疫荧光实验和蛋白印迹实验结果证实HT-1080、HUVEC、HepG2、U87Mg和PC-3细胞株CD13表达均为阳性,其中HT-1080细胞株为强阳性,而HT-29和MCF-7细胞株CD13为阴性表达.HT-1080细胞体外受体结合实验提示1h为最佳结合时间,测得Kd值为7.3 nmol/L,Bmax为0.302 nmol/L.与HT-29细胞相比,131I-NGR对HT-1080细胞株的生长抑制作用更强,并呈一定的剂量-效应和时间-效应关系；在72 h时,3700 MBq/L 131I-NGR对HT-1080的抑制率达(67.9±3.4)％.结论 经Iodogen法制备131I-NGR具有标记时间短、标记率高且标记产物稳定性好的特点.体外实验筛选出CD13表达强阳性的人肿瘤细胞株HT-1080,其与131 I-NGR结合特异性高.

abstractsObjective To synthesize 131I-NGR peptide and use it to screen CD13 expression in different tumor cell lines in vitro.Methods NGR peptide was synthesized and labeled with 131I by the Iodogen method.The radiochemical purity was evaluated with TLC.Stability was tested in double volume of normal saline and fresh human serum at room temperature and 37 ℃ at 2,12 and 24 h after labeling.Immunofluorescence and Western blot experiments were used to detect CD13 expression on HT-1080,human umbilical veins endothelial cell (HUVEC),HepG2,Ug7Mg,PC-3,HT-29 and MCF-7 tumor cell lines.Binding affinity of CD13 positive cell lines with 131I-NGR was detected by a radiochemical receptor assay.The lethal effect of various dosages of Na131I,NGR and 131I-NGR was measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on HT-1080 and HT-29 cells lines after 24,48 and 72 h incubation.The cell inhibitory rate was analyzed by one-way analysis of variance.Results NGR was successfully synthesized and labeled with 131I.Optimized labeling conditions were 18.5 MBq 131I,10 μg NGR and 20 μg Iodogen.The labeling yield of 131I-NGR was (93.7 ±2.5)％,and specific activity was 1.41 TBq/mmol.131I-NGR was stable with a radiochemical purity of more than 87％ at 24 h.Immunofluorescence experiments and Western blot demonstrated that HT-1080,HUVEC,HepG2,U87Mg and PC-3 cell lines were positive for CD13 expression,while MCF-7 and HT-29 cells lines were negative.The binding affinity of 131INGR with CD13 in HT-1080 cells was examined by a receptor binding assay and resulted in Kd =7.3 nmol/L and Bmax =0.302 nmol/L.MTT assay showed that 131I-NGR had a much stronger growth inhibitory effect on HT-1080 cells than HT-29 cells.The inhibitory rate of 131I-NGR (3700 MBq/L) on HT-1080 was (67.9 ±3.4) ％ at 72 h; while on HT-29 cells,the rate was merely (4.0 ± 0.5)％ at the same time point.Conclusions 131I-NGR can be efficiently prepared and very specifically targeted to CD13 positive human tumor cell lines.Tumor-targeted imaging and internal radiotherapy with 131I-NGR needs further research.