|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥6,200,000 (Direct Cost : ¥6,200,000)
Glomerular mesangial cells (GMC) are thought to have a pivotal role in the development of glomerular diseases. The present study was undertaken to collect the gene expression profile of GMC,a database that describes which genes are expressed and to what extent. We used a 3'-directed regional cDNA library which was constructed from cultured human GMC by cleaving cDNA with Mbo Irestriction enzyme to avoid variable cloning efficiencies reflecting the size or base composition. We randomly selected 1197 clones from the library and sequenced them with an automatic DNA sequencer. The Sequence data were compared among the clones and also with the DNA databank (NCBI). We regarded two sequences as identical when they showed more than 90 percent homology. Among the 1197 sequenced clones, 450 clones appeared more than twice (representing 83 different species) and 747 clones appeared once or twice (representing 713 different species). Among 450 redundant clones, 344 clones corresponded to 53 genes,
the functions of which are already known. When we classified these known genes according to their functions, a significant fraction of the genes expressed in human GMC are for secretable proteins (71 clones, 7 species) and for protein synthesis (74 clones, 11 species). The most abundant gene in human GMC is fibronectin (47 clones corresponding to 4.2 percent of total clones)/ A comparison of the expression profiles among different cells such as fibroblast, aortic endothelium, granulocyte, osteoblast, and HepG2, or tissues such as colon mucosa and lung, has allowed active genes to be classified as housekeepers or those with GMC cell-specific functions. We obtained 10 clones which were expressed especially in GMC,as compared to other cell types. By northern blot analysis using RNAs isolated from several human tissues, an intense signal for these clones were detected in kidney as well as cultured human GMC.One of 10 clones (GS9422) was shown to be a part of an unidentified gene with a homology to the serine protease inhibitor (serpin) family. In conclusion, the present study provide a database of genes expressed in human cultured GMC.
正常ヒト腎組織より培養メサンギウム細胞を確立した。mRNAを抽出し、HMCに発現する遺伝子解析用の3′-directed cDNAライブラリーを作製した。また、full length cDNAライブラリーも合わせて作製した。3′-directed cDNAライブラリーより任意に1200クローンを抽出しその塩基配列を決定した。その結果、蛋白合成に関与する遺伝子及び分泌蛋白の遺伝子が数多く検出された。最も発現量の多い遺伝子はファイブロネクチンであった。
上記で得られた解析結果を遺伝子データーバンク或いは他の組織(肝、肺、線維芽細胞、血管内皮細胞等)での解析結果(大阪大学細胞工学センター 大久保助教授より提供)と比較し、10個のHMC特異的且つ未知の遺伝子を選別した。これらのクローンのfull length cDNAをクローニングし解析したところ、クローンNo.9422はセリンプロテアーゼインヒビターファミリーと高い相同性を有する遺伝子であることが明らかになった。 Less