Reconstruction of basement membranes aiming at artificial organs

该研究项目的最终目标是使人工器官在上皮层和细胞外基质之间插入基底膜材料。为了在体外制造IV型胶原分子，我们做了以下研究：*不同器官中平滑肌细胞下基底膜的分子组成不同，这可能与个别器官的特定功能有关。*我们首次分离并鉴定了覆盖COL4A3和COL4A4基因5‘侧翼序列的基因组DNA片段，这为描述六种IV型胶原基因的组织特异性表达的启动子活性提供了信息。*毛囊基底膜中IV型胶原的分子形式在发育过程中是不同的。*我们通过免疫沉淀和印迹法确定了α2(IV)和α-9之间的相互作用。*当分析肿瘤进展过程中，基底膜IV型胶原分子的减少与恶性和侵袭性平行。*虽然在一只阿尔波特犬的肾脏中，α5的基因表达水平下降，但α6的基因表达水平没有变化。但这两条链的蛋白质水平都找不到。这些结果提示了α5/α6分子形式的可能性。*详细分析了IV型胶原基因在上皮基底膜中的差异表达。*在这里，我们定义了人IV型胶原的α2(IV)、α3(IV)和α6(IV)链的可溶性非胶原性结构域在调节血管生成和肿瘤生长中的新功能。这些NCI结构域被证明通过不同的αv和β1整合素依赖的机制来调节内皮细胞的黏附和迁移。*在Lmx1b(-/-)小鼠中，α3和α4在基底膜中的表达显著降低，而α1，α2和α5的表达没有变化。此外，Lmx1b特异性地与鼠/人Col4A4内含子1中的一个假定的增强子结合，并上调含有增强子样序列的报告结构。

Ultimate goal of the research project is to make artificial organs to insert basement membrane materials between epithelial layers and extracellular matrix. In order to make type IV collagen molecules in vitro, we we did the following investigations :* Molecular composition of basement membranes underneath smooth muscle cells varies in different organs, which could be related to specific function of the individual organs.* We first isolated and characterized genomic DNA fragments that cover the 5'flanking sequences of COL4A3 and COL4A4 genes, which provided information to delineate the promoter activity for the tissue-specific expression of the six type IV collagen genes.* Molecular forms of collagen IV in follicle basement membranes are different in development.* We identified interactions between the α2(IV) and ProMMP-9 by immunoprecipitations and blotting.* Basement membrane collagen IV molecules diminish in parallel with malignancy and invasiveness when analyzed in tumor progression.* Although mRNA expression level of α5 decreased in kidney of a canine Alport case, that of α6 resulted in unchanged. But the protein level of both chains cannot be found any. These results suggest a possibility of a molecular form of α5/α6.* Differential expression of collagen IV genes in epithelial basement membranes was analyzed in detail.* Here we define a novel function for soluble non-collagenous (NC1) domains of the α2(IV), α3(IV), and α6(IV) chains of human collagen type IV in the regulation of angiogenesis and tumor growth. These NCI domains were shown to regulate endothelial cell adhesion and migration by distinct αv and β1 integrin-dependent mechanisms.* In Lmx1b(-/-) mice, expression of both α3 and α4 is strongly diminished in GBM, whereas that of α1, α2 and α5 is unchanged. Moreover, LMX1B binds specifically to a putative enhancer in intron 1 of mouse/human COL4A4 and upregulates reporter constructs containing the enhancer-like sequence.