Yu F X, Gipson I K, Guo Y
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 1995 Sep;36(10):1997-2007.
The authors used and validated a recently developed method, mRNA differential display, to detect and clone genes that are differentially expressed in healing compared to stationary corneal epithelium.
RNAs from unwounded and 18-hour postwound corneal epithelia were isolated and subjected to mRNA differential display analysis. The generated cDNAs were used as probes in Northern blot analysis and in situ hybridization to confirm their differential expression and to clone longer or full-length cDNAs from a healing corneal epithelial cDNA library.
Changes in the pattern of gene expression in healing epithelium, compared with that in stationary cells, were noted. To date, 15 combinations of 5'- and 3'- primers were used with approximately 1500 mRNA species screened. Differential expression of nine mRNA species were observed. These included four known proteins. They are nonmuscle tropomyosin TM-1, cytokeratin K14, small GTP binding protein rab 11, and amyloid beta-A4 precursor-like protein-2. One is a sequence with homology to type II cytokeratin, and four represent genes with sequences that are unreported. The differential expression of five of these genes was confirmed by Northern blot analysis, in situ hybridization, or both.
mRNA differential display provides a unique and powerful experimental system to study differential gene expression in wound healing and cell migration. Using this system, differential expression of nine genes was observed. Detection of genes differentially expressed in healing epithelium may prompt studies that will define the specific role of each of the proteins in wound healing.
作者运用并验证了一种最近开发的方法——mRNA差异显示，以检测和克隆与静止角膜上皮相比在愈合过程中差异表达的基因。
从未受伤和受伤后18小时的角膜上皮中分离RNA，并进行mRNA差异显示分析。生成的cDNA用作Northern印迹分析和原位杂交的探针，以确认其差异表达，并从愈合的角膜上皮cDNA文库中克隆更长或全长的cDNA。
观察到愈合上皮中基因表达模式与静止细胞中的差异。迄今为止，使用了15种5'和3'引物组合，筛选了约1500种mRNA。观察到9种mRNA的差异表达。其中包括4种已知蛋白质。它们是非肌肉原肌球蛋白TM-1、细胞角蛋白K14、小GTP结合蛋白rab 11和淀粉样β-A4前体样蛋白-2。一种是与II型细胞角蛋白具有同源性的序列，另外4种代表序列未报道的基因。通过Northern印迹分析、原位杂交或两者证实了其中5个基因的差异表达。
mRNA差异显示为研究伤口愈合和细胞迁移中的差异基因表达提供了一个独特而强大的实验系统。使用该系统，观察到9个基因的差异表达。检测愈合上皮中差异表达的基因可能会促使开展相关研究，以明确每种蛋白质在伤口愈合中的具体作用。
