Ha Nguyen Thanh, Nakayasu Kiyoo, Murakami Akira, Ishidoh Kazumi, Kanai Atsushi
Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
Curr Eye Res. 2004 Jun;28(6):373-9. doi: 10.1080/02713680490502201.
To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray.
Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR.
Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively.
The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.
通过cDNA微阵列鉴定人圆锥角膜角膜细胞中差异表达的基因。
培养正常和圆锥角膜的角膜细胞，提取RNA。由Poly A+ RNA制备荧光染料标记的cDNA探针，与包含164个人凋亡基因的微阵列载玻片杂交，测量信号强度。使用ImaGene Ver.3.0确定圆锥角膜与正常角膜之间的表达率。通过RT-PCR和实时PCR进一步评估鉴定出的基因。
鉴定出5个过表达基因和4个低表达基因。其中，肿瘤坏死因子α诱导蛋白6（TNFAIP6）、人胰岛素样生长因子结合蛋白5（IGFBP5）和IGFBP3的差异表达通过RT-PCR得到验证和确认。实时PCR显示TNFAIP6增加了3.3倍，而IGFBP5、IGFBP3分别降低了14倍和11倍。
鉴定出的基因可能很重要，值得进一步研究。TNFAIP6、IGFBP5和IGFBP3的显著差异表达可能表明这些基因在基质变薄机制中起重要作用。
