Functional Analysis of Basement Membrane Collagen Genes

基底膜胶原基因的功能分析

• 批准号: 11694280
• 负责人: NINOMIYA Yoshifumi
• 金额: $ 5.25万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694280/
• 关键词: TYPE IV collagen; knockout mouse; supramolecular aggregates; basement membrane; vascular endothelial cells; 遺伝子発現制御

By analyzing Col4a knockout mice, it is now possible to predict the biological function of α(IV) chains in vivo. We have now knockout mice of two strains: col4a3 and col4a6. Since we have evidence that there are major three molecular forms in basement membranes: (α1)2α2, α3α4α5, and (α5)2α6, it is now possible to set-up experiments to presume in vivo function of several α chains. We also set-up international collaboration to create another new project of gene targeting for col4α1 or col4α2, which makes it possible to make mice without type IV collagen.We have solved problems of which of the 6 α(IV) chains can make molecules. We raised monoclonal antibodies specific for α(IV) chains. By using these 6 antibodies, we came to the conclusion that there are at least three molecular forms as mentioned above: (α1)2α2, α3α4α5, and (α5)2α6. New biochemical analysis using antibodies that recognize native chains made possible to immunoprecipitate specific molecules when they are digested by pure bacterial collagenase. The products run on SDS-PAGE were analyzed by other monoclonal antibodies that recognize now denatured α-chains by Western blots. This now method concluded that our prediction from immunocytochemistry was indeed true. In other words, there were three (α1)2α2, α3α4α5, and (α5)2α6 molecules. By this method, we analyzed basement membranes from renal glomeruli and bladder and aortic smooth muscle cells.Specialized basement membrane from glomeruli was analyzed to contain supramolecular aggregates of (α1)2α2 and α3α4α5 molecules. The function of it is to filter blood to create urine. The similar phenomenon is found in choroid plexus in the brain. We analyzed it and found that it contains also the same compositions as glomerulus.

通过分析Col 4a基因敲除小鼠，现在可以预测α（IV）链在体内的生物学功能。我们现在有两种基因敲除小鼠：col 4a 3和col 4a 6。由于我们有证据表明基底膜中有三种主要的分子形式：（α1）2α2，α3α4α5和（α5）2α6，现在可以建立实验来推测几种α链的体内功能。我们还建立了国际合作，创建了另一个新的col 4 α1或col 4 α2基因靶向项目，这使得制造没有IV型胶原的小鼠成为可能。我们已经解决了6 α（IV）链中的哪一个可以制造分子的问题。我们提出了α（IV）链特异性的单克隆抗体。通过使用这6种抗体，我们得出结论，至少有三种分子形式如上所述：（α1）2α2，α3α4α5和（α5）2α6。新的生化分析使用抗体，识别天然链，使免疫沉淀特定分子时，他们被纯细菌胶原酶消化。通过蛋白质印迹法，用识别变性α链的其他单克隆抗体分析SDS-PAGE上的产物。这种方法得出结论，我们从免疫细胞化学的预测确实是正确的。换句话说，有三个（α1）2α2，α3α4α5和（α5）2α6分子。用这种方法分析了肾小球、膀胱和主动脉平滑肌细胞的基底膜，发现肾小球基底膜含有（α1）2α2和α3α4α5分子的超分子聚集体。它的功能是过滤血液以产生尿液。脑内脉络丛也有类似的现象。我们分析了它，发现它也含有与肾小球相同的成分。

项目成果

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Harvey S.J. 等人：“患有 X 连锁肾炎的狗的内耳为 X 连锁阿尔波特综合征中听力损失的发病机制提供了线索”Am.J.Pathol.. 159. 1097-1104 (2001)

Kan-ju Nakano, Ken-Ichi Iyama, Junji Tsuruta, Tsuyoshi Mori, Masakazu Yoshioka, Takehisa Hiraoka, Yoshikazu Sado, and Yoshifumi Ninomiya.: "Loss of alveolar basement membrane Type IV collagen α3, α4, and α5 chains in bronchioloalveolar carcinoma of the lu

Kan-ju Nakano、Ken-Ichi Iyama、Junji Tsuruta、Tsuyoshi Mori、Masakazu Yoshioka、Takehisa Hiraoka、Yoshikazu Sado 和 Yoshifumi Ninomiya。：“细支气管肺泡癌中肺泡基底膜 IV 型胶原 α3、α4 和 α5 链的丢失卢

Morello R et al.: "Reguration of glomerular basement membrane collagen expression by LMXIB contributes to reral disease in mail pattella syndrome"Nat.Genet.. 27・2. 205-208 (2001)

Morello R 等人：“LMXIB 对肾小球基底膜胶原蛋白表达的调节导致了邮件帕特拉综合征的相关疾病”Nat Genet.. 205-208 (2001)。

Noriyuki Nagai et al.: "Localization of type IV collagen α1 to α6 chains in basement membrane during mouse molar germ development"Int.J.Dev.Biol.. 45. 827-831 (2001)

Noriyuki Nagai 等：“小鼠磨牙胚芽发育过程中基底膜中 IV 型胶原蛋白 α1 至 α6 链的定位”Int.J.Dev.Biol.. 45. 827-831 (2001)

Noriyuki Nagai, Keisuke Nakano, Yoshikazu Sado, Ichiro Naito, Mehmet Gunduz, Hidetsugu Tsujigiwa, Hitoshi Nagatsuka, Yoshifumi Ninomiya and Chong-HuAT Siar: "Localization of Type IV collagen α1 to α6 chains in basement membrane during mouse molar germ dev

Noriyuki Nagai、Keisuke Nakano、Yoshikazu Sado、Ichiro Naito、Mehmet Gunduz、Hidetsugu Tsujigiwa、Hitoshi Nagatsuka、Yoshifumi Ninomiya 和 Chong-HuAT Siar：“小鼠磨牙胚芽发育过程中基底膜中 IV 型胶原蛋白 α1 至 α6 链的定位