Establishment Of Cell Membrane Chromatography Model Of High Expression Of Insulin Receptor

OBJECTIVES: Insulin is an important peptide in human body, which has played an important role in the treatment of type I and II diabetes. But it has its deficiency as diabetes drugs. Study on small molecular compounds activating insulin receptor can overcome problems existing in production and use of insulin. CMC (cell membrane chromatography) is a new biochromatography, which integrates the separation and screening of active component and can directly screen specific components from natural products. Establishment of CMC model of high expression receptor cell lines improve specificity and efficiency of target, providing a new CMC model for anti-diabetic drug research and exploitation.METHODS: (1) Study on the specifically interaction between insulin and IR-CHO cells: HPLC determine the adsorption capacity of insulin between IR-CHO cells and CHO cells after interacting with low concentration of insulin solution and insulin of washing eluate after interacting with high concentration of insulin solution. (2) Establishment of CMC model of high expression of insulin receptor and evaluation: preparation of cell membrane of IR-CHO, immobilization of the cell membrane on silica gel carrier, filling CMSP (cell membrane stationary phase) in chromatographic column to establish the CMC model. Study on the law of the CMSP Na~+K~+-ATP enzyme activity along with time to confirm the lifetime of CMC model. Study on variation of protein content of CMSP to confirm the stability of CMC model. Study on the retain characteristics of insulin and control substance on CMC to confirm the specificity of CMC model.RESULTS: (1) HPLC determined the more adsorption capacity of IR-CHO cell than the adsorption capacity of CHO cell after interacting with concentration dose of insulin solution. (2) HPLC determined insulin from buffer (pH3.4) washing eluate of IR-CHO and did not determine insulin from buffer (pH3.4) washing eluate of CHO after interacting with high concentration of insulin solution. (3) CMSP column is quite different retain characteristics from silica column, which has the advantages of chromatographic separation and membrane activity.(4) The CMSP enzyme activity decrease along with storage time and is inversely proportional to storage temperature. (5) Insulin possesses retain characteristics on CMC model and control substance dose not act on insulin receptor in cell membrane of IR-CHO.(6) The analogy was found between the law of the retention time of insulin on the CMC varying along with time and the law of the CMSP enzyme activity varying along with time.CONCLUSIONS: Insulin can specifically interact with the cloned human insulin receptor of IR-CHO cells and the established CMC model of high expression of insulin receptor can be applied to screen IR activator, retention value is used as effecting index between ligand and receptor targets, this model is stable in the period of expiry date and can be applied to screen specific small molecule compounds activating insulin receptor.