Zhan Q, Burrows R, Cintron C
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts.
Invest Ophthalmol Vis Sci. 1995 Jan;36(1):206-15.
To develop molecular probes and to identify the cell types responsible for decorin synthesis in healing cornea.
Adult rabbit cornea and rabbit corneal stromal cell (keratocyte) culture cDNA libraries were constructed. The libraries were screened with commercially available human cDNA and oligonucleotide probes. Positive clones were sequenced to obtain a full length rabbit decorin cDNA. Synthetic oligonucleotides for rabbit decorin were chosen as probes for Northern blot analysis and in situ hybridization of healing rabbit corneas.
The cDNA sequences of the positive clones from the two libraries were identical in areas of overlap. The combined cDNA sequence indicated a 1.5-kb length with a complete open reading frame for decorin. The cDNA and deduced amino acid sequences are 90% and 88% identical, respectively, to previously reported human fibroblast and bovine bone decorin sequences. A hypervariable region near the N-terminal has little homology to decorins of other species or other rabbit protein. Northern blot analysis detected a 2.0-kb and a 2.3-kb band in mRNA from rabbit keratocyte cultures. Decorin mRNA was detected in keratocytes of normal and healing rabbit corneas by in situ hybridization. Label in the healing tissue was markedly increased above normal. Normal endothelium and epithelium in normal and healing cornea failed to show label.
Decorin mRNA from normal adult rabbit cornea is identical to decorin mRNA from keratocytes in culture and is highly homologous to decorin from other animal species. In situ hybridization indicated an upregulation of decorin message in cells adjacent to and within the healing tissue. Both stroma-derived and endothelium-derived cells in the wound synthesize message for decorin.
开发分子探针并鉴定在愈合角膜中负责核心蛋白聚糖合成的细胞类型。
构建成年兔角膜和兔角膜基质细胞（角膜细胞）培养的cDNA文库。用市售的人cDNA和寡核苷酸探针筛选文库。对阳性克隆进行测序以获得全长兔核心蛋白聚糖cDNA。选择兔核心蛋白聚糖的合成寡核苷酸作为探针，用于愈合兔角膜的Northern印迹分析和原位杂交。
两个文库中阳性克隆的cDNA序列在重叠区域相同。合并后的cDNA序列显示长度为1.5 kb，具有完整的核心蛋白聚糖开放阅读框。该cDNA和推导的氨基酸序列与先前报道的人成纤维细胞和牛骨核心蛋白聚糖序列分别有90%和88%的同一性。N端附近的一个高变区与其他物种的核心蛋白聚糖或其他兔蛋白几乎没有同源性。Northern印迹分析在兔角膜细胞培养物的mRNA中检测到2.0 kb和2.3 kb条带。通过原位杂交在正常和愈合兔角膜的角膜细胞中检测到核心蛋白聚糖mRNA。愈合组织中的标记明显高于正常水平。正常和愈合角膜中的正常内皮和上皮均未显示标记。
正常成年兔角膜的核心蛋白聚糖mRNA与培养的角膜细胞中的核心蛋白聚糖mRNA相同，并且与其他动物物种的核心蛋白聚糖高度同源。原位杂交表明愈合组织附近和内部的细胞中核心蛋白聚糖信息上调。伤口中来源于基质和内皮的细胞均合成核心蛋白聚糖的信息。
