Reconstruction of basement membranes aiming at artificial organs

针对人工器官的基底膜重建

• 批准号: 10557113
• 负责人: NINOMIYA Yoshifumi
• 金额: $ 8.26万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557113/
• 关键词: basement membrane; collagen; tissue engineering; gene expression; IV型コラーゲン; トランスフェクション; 免疫沈降; コラーゲン分子; 超分子会合体; 発現ベクター; 無細胞翻訳; 上皮細胞; リコンビナント蛋白; 高分子会合体; cDNA

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.

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：* 平滑肌细胞下的基底膜的分子组成在不同器官中不同，这可能与各个器官的特定功能有关。我们首先分离并鉴定了覆盖COL 4A 3和COL 4A 4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型胶原蛋白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鉴定了α2（IV）和ProMMP-9之间的相互作用。当分析肿瘤进展时，基底膜IV型胶原分子与恶性和侵袭性平行减少。Alport病犬肾脏α5 mRNA表达水平降低，α6 mRNA表达水平无变化。但这两条链的蛋白质水平无法找到任何。这些结果表明α5/α6可能存在一种分子形式。详细分析了上皮基底膜中IV型胶原基因的差异表达。在这里，我们定义了人IV型胶原蛋白α2（IV）、α3（IV）和α6（IV）链的可溶性非胶原（NC 1）结构域在调节血管生成和肿瘤生长中的新功能。这些NCI结构域通过不同的αv和β1整合素依赖性机制调节内皮细胞粘附和迁移。在Lmx 1b（-/-）小鼠中，GBM中α3和α4的表达均强烈减少，而α1、α2和α5的表达无变化。此外，LMX 1B特异性结合到小鼠/人COL 4A 4内含子1中的推定增强子，并上调含有增强子样序列的报告构建体。

项目成果

Saito K. et al.: "Expression of mouse α5 (IV) and α6 (IV) collagen genes in epithelial basement membranes."J.Biochem (Tokyo). 128. 427-434 (2000)

Saito K. 等人：“上皮基底膜中小鼠 α5 (IV) 和 α6 (IV) 胶原基因的表达。J.Biochem (东京) 128. 427-434 (2000)。

Nakano S. et al: "Differential tissular expression and localinzation of type IV collagen α1(IV), α2(IV) α5(IV) and α6(IV) chains and their mRNAs in normal breast, benign and malignant tumors."Laboratory Investigations. 79. 281-292 (1999)

Nakano S. 等人：“正常乳腺、良性和恶性肿瘤中 IV 型胶原 α1(IV)、α2(IV)、α5(IV) 和 α6(IV) 链及其 mRNA 的差异组织表达和定位。”实验室研究. 79. 281-292 (1999)

Petitclerc E, Boutaud A, Prestayko A, Xu J, Sado Y, Ninomiya Y, Sarras MP Jr, Hudson BG, Brooks PC.: "New Functions for Non-collagenous Domains of Human Collagen Type IV.Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo."J Biol Chem.

Petitclerc E、Boutaud A、Prestayko A、Xu J、Sado Y、Ninomiya Y、Sarras MP Jr、Hudson BG、Brooks PC.：“人类 IV 型胶原蛋白非胶原域的新功能。新型整合素配体抑制血管生成和肿瘤

Lee G E et al.: "New form of X-linked dominant hereditary nephritis in dogs"Am. J. Vet. Res... 60. 373-383 (1999)

Lee G E 等人：“犬 X 连锁显性遗传性肾炎的新形式”Am。

Tsugio Seki, Ichiro Naito, Toshitaka Oohashi, Yoshikazu Sado, and Yoshifumi Ninomiya: "Differential expression of type IV collagen isoforms, α5(IV) and α6(IV) chains, in basement membranes surrounding smooth muscle cells."Histochem Cell Biol.. 110. 359-36

Tsugio Seki、Ichiro Naito、Toshitaka Oohashi、Yoshikazu Sado 和 Yoshifumi Ninomiya：“平滑肌细胞周围基底膜中 IV 型胶原亚型、α5(IV) 和 α6(IV) 链的差异表达。”Histochem Cell Biol。 . 359-36