胰岛素-MTX受体的结合特性

The Binding Characteristics of Insulin-MTX to Insulin Receptor

目的 通过体外受体结合实验评价胰岛素 - MTX(甲氨喋呤 )的受体结合特性 ,探讨胰岛素作为受体介导靶向治疗肝癌载体的可行性。方法 将 MTX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。通过受体结合实验 ,测定胰岛素、胰岛素 - MTX的 IC50 及 Ki值 ,比较两者 IC50 及 Ki值的差异 ,以评价 MTX与胰岛素共价连接后的偶联物的受体结合特性。结果 胰岛素 - MTX能同 1 2 5I-胰岛素竞争性与肝癌细胞膜结合。胰岛素的 IC50 为 5 .0 1± 1.2 4nmol/L,Ki值为 4.85± 1.12 nm ol/L;胰岛素 - MTX的 IC50 为 93.82±19.32 nmol/L,Ki值为 91.88± 16 .86 nm ol/L。结论 胰岛素与 MTX共价偶联后 。

Objective It has been reported that several kinds of tumors express increased insulin receptor and the molecules of insulin can be internalized in cells and may thence enter into the nuclei mediated by insulin receptor. In this study, we investigated the receptor binding characteristics of insulin MTX for the possibility of using insulin as a carrier for carcinoma targeted therapy by receptor mediation. Methods MTX(methotrexate) was covalently linked to insulin directly. The insulin MTX conjugate was purified by polyacrylamine agarose gel electrophoresis and analysed by high performance liquid chromatography and SDS polyacrylamine agarose gel electrophoresis. Histologically confirmed human hepatocellular carcinoma specimens were obtained from patients at surgery and immediately frozen under -80℃. Cell membrane fractions were isolated by sucrose density gradient centrifugation. Competitive displacement of 125 I insulin with insulin and insulin MTX binding to insulin receptor were carried out and the values of IC 50 and Ki were calculated so as to observe the characteristics of insulin MTX binding to insulin receptor. Results Insulin MTX competed as effectively as insulin with 125 I insulin for insulin receptor . The values of IC 50 and Ki for insulin MTX were 93.82±19.32nmol/L and 91.88±16.86nmol/L respectively, while the values of IC 50 and Ki for insulin were 5.01±1.24nmol/L and4.85±1.12nmol/L respectively. Conclusion Insulin MTX could bind with insulin receptor with high affinity.The result demonstrates us that there is a possibility of using insulin as a carrier for carcinoma targeted therapy by receptor mediation.