Modulation of IGF bioactivity by IGF-binding proteins

胰岛素样生长因子的一个重要特征是，这些生长因子通常与其血清或培养细胞条件培养液中的特异性结合蛋白(IGFBPs)结合。已经纯化了六种不同的IGFBPs，并克隆了它们的cDNA。在此背景下，本研究旨在阐明IGFBPs对IGF生物活性的调控机制。首先，我们试图从各种培养细胞的条件培养液中纯化出每一种IGFBP。从H4-II-E细胞和BRL-3A细胞的条件培养液中，经疏水层析、阴离子交换层析和反相高效液相(C4)分离纯化得到大鼠IGFBP-1和IGFBP-2。此外，我们正在从B104s细胞中纯化大鼠IGFBP-4，方法与目前其他IGFBPs几乎相同。对于IGFBP-3、IGFBP-5或IGFBP-6，正在建立含IGFBP-3、IGFBP-5或IGFBP-6表达载体的CHO细胞系。与此同时，我们已经成功地获得了针对其中一些IGFBP的特异性抗体。将来，我们将在纯化的IGFBPs存在的情况下，用IGF-1或DES[1-3]IGF-1处理IGF-1靶细胞，然后测量IGF-1的生物活性。根据这些结果，每个内源或外源IGFBPs的生物学作用将在分子水平上得到阐明。

One of important characteristics of insulin-like growth factors is that these growth factors generally binds to their specific binding proteins (IGFBPs) in the serum or the conditioned media of cultured cells. Six distinct species of IGFBPs have been purified and their cDNAs have also been cloned. From this background, the present study is undertaken to elucidate the mechanisms of modulation of IGF bioactivity by IGFBPs. At first, we tried to purify each IGFBP from the conditioned media of various cultured cells. Rat IGFBP-1 and IGFBP-2 were purified from the conditioned media of H4-II-E cells and BRL-3A cells by the combination of hydrophobic chromatography, anion-exchange chromatography and reverse phase HPLC (C4). In addition, we are purifying rat IGFBP-4 from B104s cells in the almost same way as other IGFBPs at present. As for IGFBP-3, IGFBP-5 or IGFBP-6, establishment of the CHO cell line transfected with the expression vectors containing IGFBP-3, IGFBP-5 or IGFBP-6 cDNA is undergoing. At the same time, we have succeeded to get specific antibodies against some of these IGFBPs. In the future, we will treat IGF-1-target cells with IGF-1 or des [1-3] IGF-1 in the presence of purified IGFBPs followed by measurement of IGF-1 bioactivity. From these results, biologic roles fo each endogenous or exogenous IGFBPs will be elucidated at molecular levels.