^(125)I-VIP与小鼠肝癌H22细胞受体结合特性研究

Study of binding of ^(125)I-VIP with VIP receptor in H22 hepatocarcinoma cells in mice

目的 探讨放射性碘标记血管活性肠肽(VIP)与肝癌细胞的体内、外结合特性。方法 氯胺 T法标记、Seph adexG 5 0柱层析分离纯化制备1 2 5I VIP。通过饱和结合实验与竞争结合实验,评价1 2 5I VIP与鼠H2 2肝癌细胞受体的体外介导结合特性;进行体内动态生物分布及非标记VIP竞争分布实验,观察1 2 5I VIP在荷H2 2小鼠体内尤其在移植瘤中的分布变化规律,评价其体内受体结合特性。结果 1 2 5I标记率为73 8% ,1 2 5I VIP比活度18 2TBq mmol,放射化学纯度(RCP)98%。1 2 5I VIP与H2 2细胞受体的结合具有饱和性,Scatchard作图显示为双位点系统,高、低亲和力结合位点的解离常数(Kd)分别为1 74nmol L与2 8 63nmol L ,最大结合容量(Bmax)分别为0 2 7pmol 10 6 细胞与5 75pmol 10 6 细胞,非标记VIP以剂量依赖方式抑制1 2 5I VIP与H2 2细胞结合。各时相肿瘤组织放射性均高于肌肉(P <0 0 5 ,P <0 0 0 1) ,2 0minT NT比值达最大为2 68,肺放射性高于血液与肝脏(P <0 0 5 ,P <0 0 1) ,体内放射性主要经肾脏排泄;10 μg和40 μg非标记VIP对T NT比值的抑制率分别为2 6 87%与3 5 82 %。结论 1 2 5I VIP在体内、外与鼠H2 2肝癌细胞的结合由受体介导。此结果为进一步应用放射性核素标记VIP靶向诊治肝细胞癌研究提供了理论依据。

Objective To study the characteristics of the binding of 125 I-VIP with VIP receptor in H22 hepatocarcinoma cells in mice and in vitro. Methods VIP was radio-labelled with carrier free 125 I with the chloramine T method and purified with sephadex G-50 column chromatography. Experiments of saturation binding and competition binding were carried to study the characteristics of the binding between 125 IVIP and the VIP receptors in the H22 cells in vitro. Experiments of dynamic biodistribution and competion distribution were performed to verify the characteristics of the binding between 125 I-VIP and VIP receptors in mice with H22 hepatocarcarcinoma. Results The labelling rate of 125 I to VIP was 73.8%. The specific radioactivity of 125 I-VIP was 18.2 TBq/mmol and the radiochemical purity was 98%. The binding of 125 I-VIP with VIP receptors in H22 cells was inhibited by non-labelled VIP in a dose-dependent manner. Two classes of specific high affinity binding sites for VIP expression in H22 cells were detected with scatchard analysis. The Kd was 1.74 nmol/L and 28.63 nmol/L and Bmax was 0.27 pmol/10 6 cells and 5.57 pmol/10 6 cells for high and low affinity binding sites respectively. Radioactivity accumulation was higher in the carcinoma tissue than in the muscles of the mice bearing H22 hepatocarcinoma in each phase (P<0.05, P<0.001). The maximal ratio of the carcinoma (T)/muscle (NT) was 2.68 at 20 min p.i. The radioactivity was higher in the lungs than in the blood and the liver (P<0.05, P<0.01). The radioactivity in the mice was mainly eliminated through the kidneys. The uptake of 125 I-VIP in the carcinoma, muscles, lungs, liver and intestines was inhibited with nonlabelled VIP in a dose-dependent manner. The ratio of 10 μg and 40 μg of VIP with T/NT was 26.87% and 35.82% respectively. Conclusion The combination of 125 I-VIP with H22 hepatocarcinoma is mediated with VIP receptors of the H22 cells in vitro and in the body of mice which provides the theoretic foundation for the further study of targetting hepatocarcinoma with radionuclide-labelled VIP.