^131I-NGR的制备及人CD13表达细胞株的体外筛选

Screening CD13 expression in vitro using 131I-NGR peptide

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24h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On,0HT-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（MTF）法测定不同浓度Na^131I、NGR和^131I-NGR在24、48和72h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^131I18．5MBq、NGR10μg和Iodogen20μg，标记率为（93．7±2．5）％，比活度达1．41TBq／mmol。标记后稳定性良好，24h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，测得蚝值为7．3nmol／L，Bmax为0．302nmol／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72h时，3700MBq／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CDcr表达强阳性的人肿瘤细胞株HT-1080，其与^131I-NGR结合特异性高。

Objective To synthesize TM I-NGR peptide and use it to screen CD13 expression in dif- ferent tumor cell lines in vitro. Methods NGR peptide was synthesized and labeled with 131I by the Iodo- gen method. The radioehemical purity was evluated with TLC. Stability was tested in double volume of nor- mal saline and fresh human serum at room temperature and 37 ~C at 2, 12 and 24 h after labeling. Immuno- fluorescence and Western blot experiments were used to detect CDI3 expression on HT-1080, human umbili- cal veins endothelial cell （HUVEC） , HepG2, U87Mg, PC-3, HT-29 and MCF-7 tumor cell lines. Binding affinity of CD13 positive cell lines with 1311-NGR was detected by a radiochemica] receptor assay. The lethal effect of various dosages of NaTM I, NGR and 1311-NGR was measured by an 3-（4,5-dimethyhhiazol-2-yl） -2, 5-diphenyhetrazolium bromide （MTr） assay on HT-1080 and HT-29 cells lines after 24, 48 and 72 h incu- bation. The cell inhibitory rate was analyzed by one-way analysis of variance. Results NGR was success- fully synthesized and labeled with 131I. Optimized labeling conditions were 18.5 MBq 131I, 10 μg NGR and 20 Ixg 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 experi- ments 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 ceils lines were negative. The binding affinity of 131I- NGR with CD13 in HT-1080 cells was examined by a receptor binding assay and resulted in Kd = 7.3 nmol/L and Bin= = 0. 302 nmol/L. MTT assay showed that 131I-NGR had a much stronger growth inhibitory effect on HT-1080 ceils 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. Con- clusions 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.