瘢痕疙瘩和正常皮肤组织中基因表达的变化

Analysis of differentially expressed genes in keloids and normal control skin

目的 应用基因表达谱芯片筛选瘢痕疙瘩发生相关基因 ,并对部分基因的功能进行初步分析。方法 按微阵列排列 ,将84 0 0种人类基因PCR产物制成微阵矩表达谱芯片 ;提取瘢痕疙瘩和正常皮肤的总RNA ,并纯化mRNA。将等量的两种组织的mRNA分别进行逆转录 ,合成荧光分子(Cy5或Cy3)掺入的cDNA作为探针 ,芯片杂交和严格洗片后 ,扫描芯片荧光信号图像 ,计算机分析比较两种组织中差异表达的基因。通过RT PCR方法检测NGF、TGF β1和c myc基因在不同组织中的表达变化。结果 在所检测的 3对临床标本中 ,瘢痕疙瘩和正常皮肤间存在差异表达的基因有 4 0 2条(4 75 % ) ,主要涉及胞外基质、运输蛋白、细胞信号和传导蛋白、细胞骨架和运动相关蛋白的基因等。这些差异表达基因与瘢痕疙瘩的发生可能存在相关性。RT PCR研究发现 ,瘢痕疙瘩内NGF、TGF β1和c myc的基因表达水平均明显高于正常皮肤。结论 多种基因参与调控瘢痕疙瘩的发生 ,对相关基因群的研究有助于认识瘢痕疙瘩的形成机制。NGF、TGF β1和c myc在瘢痕疙瘩形成过程中可能起着重要的调节作用。

Objective To study the differentially expressed genes between keloids and normal control skin using cDNA microarray. Methods The PCR products of 8400 human genes were spotted on a chemical-material-coated-glass plate in array. Total RNAs were isolated from the freshly excised human keloids and normal control skin,and then were purified to mRNAs,which were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP,for preparing the hybridization probes. The mixed probes were hybridized to the cDNA microarray. According to the results of cDNA microarray,three genes (NGF,TGF-β_ 1 and c-myc) were chosen to study their differential expression in keloids and normal control skin by RT-PCR. Results Among the 8400 target genes,there were 402 genes with different expression (4.75%),and they were mainly associated with extracellular matrix genes,cellular signal molecule genes and cellular skeleton molecule genes. Analysis of collagen related molecule and growth factor gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. RT-PCR showed that the levels of gene expression of NGF,TGF-β_ 1 and c-myc were all higher in keloids than those in normal control skin. Conclusion Many genes were found to be involved in the formation of keloids. Further analysis of the obtained genes might contribute to reveal the molecular mechanism of keloids formation,in which NGF,TGF-β_ 1 and c-myc might play important roles.