MULTIPLE ANALYSIS OF PERIODONTAL LESIONS BY DIFFERENTIAL DISPLAY

本研究旨在利用基因芯片技术研究牙周组织破坏相关炎性细胞因子、生长因子和酶的大规模基因表达，为牙周病的诊断提供依据。我们选择了约100个已知基因作为排列的靶点，其中包含与牙周病发病密切相关的炎性细胞因子、生长因子和酶。根据人牙周膜(PDL)基因表达谱信息，我们从PDL的3‘cDNA文库中添加了约1100个非冗余克隆用于排列靶点。用聚合酶链式反应扩增靶DNA，用微阵列技术将约1,200种cDNA印在涂有聚赖氨酸的玻片上。为了验证CDNA基因芯片在牙周病诊断中的应用，我们从不同时间点培养的牙周膜细胞中提取RNA，逆转录后用Cy3和Cy5差异标记，杂交到芯片上。因此，我们检测了几个基因在PDL细胞分化过程中的差异表达。此外，我们还将该基因芯片应用于牙周组织的大规模基因表达分析。从牙周手术中采集的牙周组织提取总RNA，体外培养人牙周成纤维细胞(HGF)。这些RNA被逆转录，用Cy3和Cy5差异标记，并杂交到芯片上。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，在肝细胞生长因子中检测到胶原和细胞黏附分子基因的上调。结果表明，本研究建立的cDNA微阵列是分析牙周组织中大规模基因表达的有力工具，提示利用该cDNA对牙周疾病状况进行多种分析是可能的。

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