Garcia A Minerva, Szasz Nora, Trippel Stephen B, Morales Teresa I, Grodzinsky Alan J, Frank Eliot H
Continuum Electromechanics Group, Center for Biomedical Engineering, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Arch Biochem Biophys. 2003 Jul 1;415(1):69-79. doi: 10.1016/s0003-9861(03)00215-7.
This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion-reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.
本研究聚焦于胰岛素样生长因子（IGF）结合蛋白（IGFBPs）在软骨组织中对IGF-I的转运及结合作用。我们已开发出实验和理论建模技术，以量化并对比扩散、结合、流体对流及电迁移在IGF-I于软骨组织内转运过程中的作用。将牛关节软骨圆盘置于含有125I-IGF-I及不同浓度未标记IGF-I的缓冲液中进行平衡处理。通过测量组织中125I-IGF-I（游离加结合态）的摄取率与缓冲液中标记物浓度之比来测定平衡结合情况，结果发现其与涉及基质内一个主要结合位点家族的一级可逆结合模型相符。蛋白质免疫印迹法显示在约23 kDa处有一个主要的IGF结合双峰，先前已证明其与IGFBP-6一致。测定了125I-IGF-I在软骨中的扩散转运，发现其与纳入一级可逆结合的扩散限制反应理论模型相符。在125I-IGF-I稳态转运过程中加入过量未标记IGF-I，如扩散反应模型所预测的那样，导致结合的125I-IGF-I从组织中释放。相比之下，加入低亲和力的Des(1-3)IGF-I类似物并未导致结合的125I-IGF-I释放。施加电流通过电渗和电泳来增强IGF-I在软骨中的转运。综合来看，我们的结果表明，一个单一的主要底物家族，即高亲和力IGFBPs，对软骨中观察到的大部分IGF-I结合负责。数据表明，组织内液流，如体内软骨机械加载所诱导的液流，可能会使IGF转运增强一个数量级，并且这种增加可能有助于抵消结合蛋白对IGF固定所造成的限制。
