Isolation of a gene product expressed by a subpopulation of human lung fibroblasts by differential display
Isolation of a gene product expressed by a subpopulation of human lung fibroblasts by differential display
复制标题
DOI：
10.1165/ajrcmb.20.2.3368
复制
发表时间：
1999-02-01
影响因子：
6.4
通讯作者：
Narayanan, AS
中科院分区：
文献类型：
--
作者：
Lurton, J;Rose, TM;Narayanan, AS
成纤维细胞是负责合成肺和其他结缔组织中基质成分的主要细胞类型。有证据表明，成纤维细胞是异质性的，并且在纤维化疾病中具有一些不同性质的亚群被克隆选择和扩增。然而，迄今为止，很少有能够证明组织中成纤维细胞亚群存在的独特标记物被分离出来。为了确定能够检测成纤维细胞亚群的蛋白质，我们通过差异显示比较了两种培养的人肺成纤维细胞亚群的信使RNA（mRNA）表达。获得总RNA，合成互补DNA（cDNA），并比较用几对引物获得的聚合酶链反应（PCR）产物。鉴定并克隆了一个724 bp的产物，该产物在一个人肺成纤维细胞亚群中强烈表达。该产物在其他肺成纤维细胞亚群中表达较差。编码该产物的基因的mRNA在人平滑肌细胞、内皮细胞或上皮细胞中未检测到，尽管其存在于真皮成纤维细胞中。在正常和纤维化的人肺中检测到mRNA。用从该克隆获得的序列搜索国家生物技术中心（NCBI）GenBank DNA数据库，没有发现显著匹配。然而，对表达序列标签（dBEST）的NCBI数据库的搜索揭示了对应于LR8 cDNA序列的5个不同的人表达序列标签（EST）克隆。另外6个小鼠和1个猪EST克隆被鉴定出与人成纤维细胞cDNA具有显著的相似性。人成纤维细胞基因产物和小鼠同源物的整个编码序列的复合物由各自的重叠EST序列组装而成。为每个复合序列鉴定的开放阅读框分别预测人和小鼠序列的270和263个氨基酸的蛋白质产物，其具有52%的同一性，具有三个缺口。在氨基酸水平上，在NCBI DNA和蛋白质数据库或Blocks数据库的穷举搜索中未检测到与任何其他序列的显著序列相似性。通过使用LR8上游的正义引物和LR8内的反义引物获得具有预测长度和序列的PCR产物。我们的研究结果表明，这种差异显示的产品代表了以前未描述的蛋白质，可能是有用的区分成纤维细胞，可能成纤维细胞亚群，从其他细胞类型在肺和其他组织。
Fibroblasts are the major cell type responsible for synthesizing matrix constituents in lung and other connective tissues. Evidence indicates that fibroblasts are heterogeneous, and that subpopulations with some distinct properties are clonally selected and expanded in fibrotic diseases. However, few distinct markers capable of demonstrating the presence of fibroblast subpopulations in tissues have been isolated so far. With the objective of identifying proteins that could detect fibroblast subpopulations, we compared the messenger RNA (mRNA) expression of two cultured human lung fibroblast subpopulations by differential display. Total RNA was obtained, complementary DNA (cDNA) was synthesized, and the polymerase chain reaction (PCR) products obtained with several primer pairs were compared. One 724-bp product, which was strongly expressed by one human lung fibroblast subpopulation, was identified and cloned. This product was poorly expressed by the other lung fibroblast subpopulation. The mRNA for the gene encoding this product was not detectable in human smooth-muscle cells, endothelial cells, or epithelial cells, although it was present in dermal fibroblasts. The mRNA was detected in normal and fibrotic human lungs. Search of the National Center for Biotechnology (NCBI) GenBank DNA database with the sequence obtained from this clone revealed no significant matches. However, a search of the NCBI database of expressed sequence tags (dBEST) revealed five different human expressed sequence tag (EST) clones corresponding to the LR8 cDNA sequence. Six additional mouse and one pig EST clones were identified that showed significant similarity to the human fibroblast cDNA. Composites of the entire coding sequences for the human fibroblast gene product and the mouse homologue were assembled from the respective overlapping EST sequences. The open reading frame identified for each composite sequence predicted protein products of 270 and 263 amino acids for the human and mouse sequences, respectively, which were 52% identical, with three gaps. At the amino acid level, no significant sequence similarity was detected with any other sequences in exhaustive searches of the NCBI DNA and protein databases or the Blocks databases. A PCR product with predicted length and sequence was obtained by using a sense primer upstream to LR8 and an antisense primer within LR8. Our results indicate that this differentially displayed product represents a previously undescribed protein that could be useful for distinguishing fibroblasts, and possibly fibroblast subpopulations, from other cell types in lungs and other tissues.