Application of a new cell-adhesive and cell-migatory molecule, laminin-5B, to regenerative medicine

层粘连蛋白是一类细胞外基质蛋白家族，主要定位于基底膜，调节多种细胞功能，如黏附、运动、增殖、凋亡和分化。到目前为止，已经鉴定了超过15种由这三条链的不同组合组成的层粘连蛋白亚型。其中，层粘连蛋白-5(α3A/B、β3、γ2；层粘连蛋白-332)强烈促进细胞的黏附和迁移。最近，我们建立了人层粘连蛋白-5A(αA，β3，γ2)和层粘连蛋白-5B(α3B，β3，γ2)的高效表达系统。在这项研究中，我们研究了将这些层粘连蛋白用于再生医学的可能性。我们还试图建立一种新的层粘连蛋白异构体-层粘连蛋白-6B(α3B，β1，γ1)的制备体系。主要研究结果如下。1)间充质干细胞(MSC)被认为是再生医学中一种重要的细胞类型，因为它可以分化为成骨细胞、软骨细胞、神经细胞、肌肉细胞等。我们发现层粘连蛋白-5A和层粘连蛋白-5B有效地刺激了培养的人MSCs的生长，同时抑制了其向软骨方向的分化。这提示，层粘连蛋白-5A/B有可能作为一种促进生长的补充剂来促进人MSCs的体外高效生长。2)预测了层粘连蛋白-6B的存在，但从未被证实。我们发现这种层粘连蛋白存在于人体各种组织的微血管基底膜中。我们还通过将人层粘连蛋白α3B、β1、γ1链的cDNA导入HEK293细胞，建立了该新型层粘连蛋白的高效表达系统。(α3A、β3、γ2)。这种层粘连蛋白可能在血管生成和维持血管结构和功能中发挥重要作用。

Laminins are a family of ECM proteins that are localized mainly in basement membranes and regulate various cellular functions such as adhesion, motility, proliferation, apoptosis and differentiation. More than 15 laminin isoforms consisting of different combinations of the three chains have been identified thus far. Among them, laminin-5 (α3A/B, β3, γ2 ; laminin-332), strongly promotes cellular adhesion and migration. Recently we established efficient expression systems of human recombinant laminin-5A (αA, β3, γ2) and laminin-5B (α3B, β3, γ2). In this study, we investigated the possibility to use these laminins for regenerative medicine. We also attempted to establish a system to produce a novel laminin isoform, laminin-6B (α3B, β1, γ1). Main results are as follows. 1) Mesenchymal stem cell (MSC) is thought to be an important cell type in the regenerative medicine, because it can differentiate into osteoblasts, chondrocytes, neural cells, muscle cells and some others. We found that laminin-5A and laminin-5B efficiently stimulated the growth of human MSCs in culture, while suppressing their chondrogenic differentiation. This suggest the possible use of laminin-5A/B as a growth-stimulatory supplement for the efficient in vitro growth of human MSCs. 2) The presence of laminin-6B is predicted, but it has never been identified. We found that this laminin existed in the basement membranes of micro-blood vessels in various human tissues. We also established an efficient expression system of this novel laminin by introducing cDNAs for human laminin α3B, β1, γ1 chains into HEK293 cells. (α3A, β3, γ2). This laminin may play an important role in angiogenesis and maintenance of vascular structures and functions.