Biological Function of Type VIII Collagen

为了了解哪些细胞或器官产生VIII型胶原，我们从各种细胞和组织中提取RNA，并使用特定cDNA进行北方印迹分析。发现培养的人主动脉内皮细胞持续合成α 1（VIII）和α 2（VIII）链，直到第10代。这些细胞同时合成I型、III型和V型胶原。在单独的实验中，测试大鼠主动脉平滑肌细胞是否产生胶原蛋白。我们检测到的是细胞合成75 kDa的细菌胶原酶敏感物质。感兴趣的是，通过向培养基中添加肝素，该材料的产量提高了20至30倍。目前，我们正在通过北方印迹分析来测试这种细菌胶原酶敏感材料是否是VIII型的α链。Northern印迹和原位杂交结果表明，小鼠角质形成细胞在mRNA水平上表达VIII型胶原，几种转基因小鼠在发育过程中VIII型胶原基因的表达模式存在差异。它的模式似乎是严格的和发展的规定。然而，我们不知道该模式是人为的还是真实的，因为我们没有预测表达模式;在某些组织中是短暂的。用小鼠VIII型胶原特异性探针进行了直接原位杂交，证明了其表达模式是真实的。

In order to know which cells or organs produce type VIII collagen, we extracted RNA from various cells and tissues and performed Northern blot analysis using specific cDNAs. Cultured human aortic endothelial cells were found to keep synthesizing alpha1 (VIII) and alpha2 (VIII) chains until tenth passage. These cells synthesize collagen type I,III,and V at the same time. In separate experiment rat aortic smooth muscle cells were tested whether they produce collagens. What we detected was that the cells synthesized 75 kDa bacterial collagenase sensitive materials. Of interest was that production of this materials was enhanced 20 to 30 times by addition of heparin into the culture medium. Currently we are testing whether this bacterial collagenase sensitive material is the alpha-chain of type VIII by Northern blotting analysis. We also demonstrated that mouse keratinocytes produce collagen VIII at the mRNA level using Northern-blotting and in situ hybridization.Several lines of transgenic mice did show strange expression pattern of type VIII collagen gene during development. It seemed that its pattern was strict and developmentally regulated. However, we did not know whether the pattern was artifact or real, since we did not predict the expression pattern ; transient in certain tissues. Direct in situ hybridization was performed using the specific probes for mouse collagen type VIII at the developmental stage and proved that the expression pattern was real.