甲基乙二醛诱导牙周膜成纤维细胞基因表达与分析

Gene Expression of Human Periodontal Ligament Fibroblasts Induced by Methylglyoxal Using cDNA Microarray and Bioinformatics Analysis

运用基因芯片研究甲基乙二醛诱导人牙周膜成纤维细胞基因表达谱的变化。原代培养人牙周膜成纤维细胞,诱导组以终质量浓度为0.1 g/L的甲基乙二醛刺激培养细胞,对照组不含甲基乙二醛。24 h后收获细胞,提取mRNA,逆转录cDNA时用Cy3和Cy5荧光染料标记,制备成cDNA探针,与表达谱芯片进行杂交、扫描和分析。芯片检测结果用实时定量聚合酶链反应验证和生物信息学分析。结果共有18条基因显著差异表达,其中上调基因有11条,下调基因有7条,差异性表达的基因按功能可分为程序性细胞死亡、信号转导、细胞因子、代谢酶类、载体蛋白和未知基因等。与程序性细胞死亡、信号转导和细胞因子相关基因的差异表达可能是甲基乙二醛通过线粒体信号通路,诱导人牙周膜成纤维程序性细胞死亡,破坏牙周组织增生,从而导致牙周病发生的机制。

To investigate the gene expression of human periodontal ligament fibroblasts（HPDLF） induced by methylglyoxal used cDNA microarray analysis.HPDLF were primary cultured with methylglyoxal-induced（MG1,MG2） or methylglyoxal free（C1,C2）,which were used to screen the related genes by microarray technology.Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes were prepared from the mRNA samples of induced group（MG1,MG2） and control group（C1,C2） cells by reverse transcription method.The two color probes were mixed and hybridized on the cDNA chip.Differential expressed genes of the two groups were analyzed using computer.Then the differently expressed genes were further validated by real-time PCR technique and bioinformatics analysis.There were 18 genes showed significantly different expression between induced group and control group.Among the 18 genes,11 were up regulated and 7 were down.The results of real-time PCR analysis for the differently expressed gene were coincident with those of microarray assay.The results indicated that regulating the genes expression may be the mechanism of apoptosis of HPDLF induced by MG and etiology of periodontitis through mitochondria signal pathway,involved in differential expressed genes of signal transduction,apoptosis,cytokine,metabolic enzyme etc.