MULTIPLE ANALYSIS OF PERIODONTAL LESIONS BY DIFFERENTIAL DISPLAY

本研究旨在利用CDNA芯片研究炎症因子、生长因子和牙周组织破坏相关酶的大规模基因表达在牙周病诊断中的应用。我们选择了大约100个已知的基因来排列靶标，这些基因含有与牙周病发病密切相关的炎症因子、生长因子和酶。基于人类牙周韧带（PDL）基因表达谱的信息，我们从PDL的3’cdna文库中添加了约1100个非冗余克隆。采用PCR扩增靶基因，利用微阵列将约1200种cDNA打印到聚赖氨酸涂膜玻片上。为了验证CDNA芯片对牙周病的诊断价值，我们从PDL中分离出RNA。将不同时间点培养的细胞和rna进行反转录，用Cy3和Cy5进行差异标记，并将其杂交到芯片上。结果，我们检测到PDL细胞分化过程中几个基因的差异表达。此外，我们将这种cDNA芯片应用于牙周组织的大规模基因表达分析。从牙周手术牙龈组织和体外培养的人牙龈成纤维细胞（HGF）中提取总RNA。将rna逆转录并用Cy3和Cy5进行差异标记，并将其杂交到微阵列上。在牙龈组织中，我们检测到涉及免疫活性细胞的基因表达上调，如免疫球蛋白基因和趋化因子基因。另一方面，在HGF中检测到胶原蛋白和细胞粘附分子基因的上调。目前的研究结果表明，本研究开发的cDNA微阵列是分析牙周组织中大规模基因表达的有力工具，并表明利用该cDNA进行牙周疾病状况的多重分析是可能的

This study was designed to investigate the largescale gene expression of inflammatory cytokines, growth factors and enzymes involved periodontal tissue destruction by CDNA microarray for diagnosis of periodontal diseases. We selected about 100 known genes for arrayed targets, contained inflammatory cytokines, growth factors and enzymes these were closely related with pathogenesis of periodontal diseases. Based on the information of the gene expression profile of human periodontal ligament (PDL), we added about 1,100 nonredundant clones for arrayed targets from 3'cDNA library ofPDL. PCR was performed to amplify cDNAs for the targets and about 1,200 species of cDNA were printed onto a polyLlysine coated slide glass using the microarrayer. In order to verify the CDNA microarray for diagnosis of eriodontal diseases, we isolated RNA from PDL.cells cultured at different time points and RNAs were reversetranscribed and differentially labeled with Cy3 and Cy5, and hybridized onto the microarray. In result, we detected the differential expression ofseveral genes during the cytodifferentiation of PDL cells. Furthermore, we applied this cDNA microarray to the largescale gene expression analysis in periodontal tissue. Total RNA was extracted from the gingival tissue sampled at periodontal surgery and human gingival fibrobrast (HGF) cultured in vitro. The RNAs were reverse transcribed and differentially labeled with Cy3 and Cy5, and hybridized onto the microarray. In the gingival tissue, we detected upregulation of the gene expressions involved immunocompetent cells, such as immunogloburin genes and chemokine genes. In another way, the upregulation of the genes of collagen and cell adhesion molecules was detected in HGF. Present results show that the cDNA microarray developed in this study is a powerful tool to analyze the largescale gene expression in periodontal tissue and suggest that there is a possibility of multiple analysis for periodontal disease condition by this CDNA