常染色体显性多囊肾组织差异表达基因的初步研究

Detection of differentially expressed genes in human autosomal dominant polycystic kidney tissue

目的应用基因芯片技术及最新公共数据库,筛选常染色体显性多囊肾组织中差异表达的基因,对其进行功能分类,并对其中1条基因利用原位杂交技术进行验证。方法将代表8398条人类基因的PCR产物制成基因芯片。将等量的多囊肾组织和正常肾组织mRNA分别用Cy5、Cy3荧光标记,逆转录合成cDNA探针,混合后与上述基因芯片杂交。扫描杂交信号荧光强度,找出差异表达基因,对获得的基因进行分子生物信息学分析。并对其中的上调表达基因IGF1mRNA进行原位杂交,验证基因芯片结果的准确性。结果(1)在进入研究的8398条基因中,共发现357条差异表达基因。94条基因在多囊肾组织中低表达,263条基因高表达;(2)上调表达基因主要属于原癌基因,细胞骨架蛋白和运动相关蛋白,凋亡相关蛋白,细胞信号和传递蛋白,细胞因子;下调表达基因主要属于抑癌基因,DNA结合、转录和转录因子,细胞信号和传递蛋白,参与代谢的基因;(3)IGF1mRNA原位杂交结果与芯片结果一致。结论基因表达谱芯片可快速、高效地筛选差异表达基因;多囊肾病的发生、发展中存在着多种不同功能基因表达调控的改变。

Objective To detect the differentially expressed genes in human polycystic kidney by cDNA microarray. Methods The PCR products of 8398 genes were spotted onto a chip in array. Both mRNAs isolated from polycystic kidney tissue and normal kidney tissue were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP （ Cy5-dUTP and Cy3-dUTP） for preparing the hybridization probes. The mixed probes were hybridized to the cDNA microarray. Then the cDNA microarray was scanned for the fluorescent signals and the display of differences between the 2 tissues. IGF1 mRNA, one of the up regulated genes was detected by in situ hybridization technique in the two tissues to validate the result from cDNA microarray. Results The result indicated that the expressions of 263 genes were up regulated while the expressions of 94 genes were down regulated in the polycystic kidney tissue among the 8598 target genes. Bioinformatical analysis of those genes had been performed. The up-regulated genes were mainly the ones of oncogene, cellular skeleton and movement, apoptosis related protein, cell signal transduction protein, and cytokine. The down regulated genes were mainly the ones of anti-oncogene, DNA binding and transcription factors, cell signal transduction protein, and metabolism protein. The IGF1 mRNA expression detected by in situ hybridization was consequently consistent with the cDNA microarray. Conclusion cDNA microarray is an effective and quick method for studying differential expressed genes. Three hundred and fifty-seven differentially expressed genes with different functions were revealed in the polycystic kidney tissue, which may play some roles in the progression of polycystic kidney.