Functional characterization of Laminin 10

层粘连蛋白 10 的功能表征

• 批准号: 5244254
• 负责人: Professorin Dr. Lydia Sorokin
• 金额: --
• 依托单位: Institut für Physiologische Chemie und Pathobiochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2000
• 资助国家: 德国
• 起止时间: 1999-12-31 至 2002-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5244254?language=en
• 关键词: Functional; characterization; Laminin; 10

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.

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 （alpha4 beta1 gamma1）在所有内皮细胞中广泛表达，与发育阶段无关，而层粘连蛋白10 （alpha5 beta1 gamma1）仅限于成熟毛细血管基底膜（BM）和一些小静脉，仅在出生后3 - 4周出现。内皮细胞的激活状态进一步调节层粘连蛋白α 4和α 5链的表达；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以及为什么它们在这些细胞中受到不同的调节尚不清楚，但表明了功能上的区别。最近，层粘连蛋白α - 5链在小鼠体内被清除，然而，动物在胚胎发育中过早死亡，无法识别具有重要功能的位点。因此，我们建议使用Cre-loxP重组酶系统，在内皮细胞特异性启动子（Tie-1）的控制下表达Cre-loxP，以消除内皮细胞BM特异性表达的层粘连蛋白alpha5，以评估其在该位点的功能。此外，将使用Cre- loxp重组酶以一种时间依赖性的方式减少层粘连蛋白alpha5的表达，Cre在诱导启动子（修饰的雌激素受体-他莫昔芬系统）的控制下。出生后3 - 4周，当层粘连蛋白α 5首次出现在内皮细胞骨髓中时，其表达将被消除。我们实验室进行的原位杂交研究表明，除了上皮细胞外，出生后内皮细胞以外的层粘胶蛋白α 5 mRNA的表达很少（28,30）。因此，该系统不仅提供了检测层粘连蛋白α 5在内皮细胞中的功能的机会，也提供了检测成熟上皮细胞中层粘连蛋白α 5功能的机会。