Functional characterization of Laminin 10

我们已经鉴定并鉴定了内皮细胞表达的层粘连蛋白亚型;层粘连蛋白8（α 4 β 1 γ 1）广泛表达于所有内皮细胞，而与发育阶段无关，而层粘连蛋白10（α 5 β 1 γ 1）仅限于成熟毛细血管和一些小静脉的基底膜（BM），仅在出生后3 - 4周出现。层粘连蛋白α 4和α 5链的表达进一步受内皮细胞活化状态的调节;在炎症事件中起作用的细胞因子如IL-1和TNF-α上调层粘连蛋白α 4;而血管生成抑制剂如孕酮及其衍生物上调层粘连蛋白α 5。为什么不同的血管表达不同的层粘连蛋白，为什么他们在这些细胞中的差异调节是未知的，但表明功能的差异。层粘连蛋白α 5链最近已在小鼠中被消除，然而，动物在胚胎发生中过早死亡，无法鉴定具有功能意义的位点。因此，建议使用Cre-表达在内皮细胞特异性启动子（Tie-1）控制下的Cre-loxP重组酶系统，以从内皮细胞BM特异性消除层粘连蛋白α 5表达，从而评估其在该位点的功能。此外，层粘连蛋白α 5表达将使用Cre-loxP重组酶以时间依赖性方式消除，其中Cre在诱导型启动子的控制下（修饰的雌激素受体-他莫昔芬系统）。当层粘连蛋白α 5首次出现在内皮BM中时，其表达将在出生后3 - 4周消除。我们实验室进行的原位杂交研究表明，出生后，除上皮细胞外，层粘连蛋白α 5 mRNA在内皮细胞以外的其他部位的表达最低（28，30）。因此，该系统不仅提供了检查内皮中层粘连蛋白α 5功能的机会，而且还提供了检查成熟上皮中层粘连蛋白α 5功能的机会。

We have identified and characterized the laminin isoforms expressed by endothelium; laminin 8 (alpha4 beta1 gamma1) is widely expressed in all endothelium regardless of the stage of development, while laminin 10 (alpha5 beta1 gamma1) is restricted to basement membranes (BM) of mature capillaries and some venules, appearing only 3 - 4 weeks after birth. Expression of laminin alpha4 and alpha5 chains is further regulated by the activation state of endothelial cells; cytokines which play a role in inflammatory events such as IL-1 and TNF-alpha upregulate laminin alpha4; while angiostatic agents such as progesterone and its derivatives upregulated laminin alpha5. Why different vessels express different laminins and why they are differentially regulated in these cells is unknown, but suggests functional distinction. The laminin alpha5 chain has recently been eliminated in mice, however, the animals die too early in embryogenesis to permit identifications of sites of functional significance. It is proposed, therefore, to use the Cre-loxP recombinase system, with Cre-expression under the control of an endothelial cell specific promoter (Tie-1), to eliminate laminin alpha5 expression specifically from endothelial cell BM to assess its function at this site. Further, laminin alpha5 expression will be ablated in a time-dependent manner using Cre-loxP recombinase, with Cre under the control of an inducible promoter (the modified oestrogen receptor - tamoxifen system). Laminin alpha5 expression will be eliminated 3 - 4 weeks after birth when it first appears in endothelial BM. In situ hybridization studies carried out in our laboratory have shown that laminin alpha5 mRNA expression at sites other than endothelium is minimal after birth, with the exception of epithelia (28,30). This system therefore privides not only the opportunity of examining the function of laminin alpha5 in endothelium, but also in mature epithelium.