胰岛素-IUdR受体结合特性研究

Study on insulin receptor binding characteristics of insulin-IUdR

目的 探讨胰岛素作为受体介导靶向治疗肝癌药物载体的可行性。方法 将碘苷 (I UdR)与胰岛素共价连接 ,采用聚丙烯酰胺凝胶电泳分离纯化 ,高效液相层析鉴定 ,SDS 聚丙烯酰胺凝胶电泳测定相对分子质量。通过受体结合实验 ,分别测定胰岛素、胰岛素 IUdR的IC50 及KI值 ,评价IUdR与胰岛素共价连接后的受体结合特性。结果 胰岛素 IUdR能同12 5I 胰岛素竞争性与肝癌细胞膜结合。胰岛素的IC50 1为 (5 .0 1± 1.2 4)nmol/L ,IC50 2为 (17.75± 4.86 )nmol/L ,KI1值为 (4 .85±1 12 )nmol/L ,KI2为 (17.6 9± 4.81)nmol/L ;胰岛素 IUdR的IC50 1为 (11.5 0± 2 .83)nmol/L ,IC50 2为(19 35± 5 .11)nmol/L ,KI1值为 (11.2 6± 2 .6 5 )nmol/L ,KI2为 (19.30± 5 .0 2 )nmol/L。结论 胰岛素与IUdR共价偶联后 ,仍具有与胰岛素受体结合的生物活性。

Objective It has been reported that several kinds of tumors express increased insulin receptors and the molecule of insulin can be internalized in cells and enter the nuclei through the mediation of the insulin receptor.In this study,we investigated the receptor binding characteristics of insulin-IUdR for the possibility of using insulin as carriers for carcinoma targeted therapy by receptor mediation. Methods IUdR (idoxuridine) was covalently linked to insulin using the bifunctional reagent succinic acid. The insulin-IUdR conjugate was purified by polyacrylamide agarose gel electrophoresis and analysed by high performance liquid chromatography and SDS-polyacrylamide agarose gel electrophoresis. The human hepatocellular carcinoma specimens confirmed histologically were obtained from patients at surgery and immediately frozen under -80 ℃. Cell membrane fractions were isolated by sucrose density gradient centrifugation.Competitive displacement of 125I-insulin with insulin and insulin-IUdR binding to the insulin receptor were carried out. Values of IC 50 and KI were calculated to observe the characteristics of insulin-IUdR binding to the insulin receptor. Results Insulin-IUdR competed as effectively as insulin with 125I-insulin for binding to insulin receptors. The values of IC 501 and IC 502 for insulin-IUdR were (11.50±2.83) nmol/L and (19.35±5.11) nmol/L, respectively; the values of KI1 and KI2 for insulin-IUdR were (11.26±2.65) nmol/L and (19.30±5.02) nmol/L, respectively, while values of IC 501 and IC 502 for insulin were (5.01±1.24) nmol/L and (17.75±4.86) nmol/L, respectively. The values of KI1 and KI2 for insulin were (4.85±1.12) nmol/L and (17.69±4.81) nmol/L, respectively. Conclusions Insulin-IUdR could bind with the insulin receptor with high affinity.The result shows that there is a possibility of using insulin as carriers for carcinoma targeted therapy through receptor mediation.