MULTIPLE ANALYSIS OF PERIODONTAL LESIONS BY DIFFERENTIAL DISPLAY

本研究应用基因芯片技术检测牙周组织破坏相关的炎症因子、生长因子和酶的大规模基因表达，为牙周病的诊断提供实验依据。我们选择了100多个已知的基因作为阵列靶点，包括与牙周病发病密切相关的炎症因子、生长因子和酶。根据人牙周膜（PDL）基因表达谱的信息，我们从PDL 3 '端cDNA文库中添加了约1,100个非冗余克隆作为阵列靶点。进行PCR以扩增靶的cDNA，并使用微阵列仪将约1，200种cDNA打印到聚赖氨酸涂覆的载玻片上。为了验证cDNA微阵列在诊断碘缺乏病中的应用，我们从培养不同时间的PDL细胞中提取RNA，反转录后用Cy 3和Cy 5进行差异标记，并与基因芯片杂交。结果表明，在PDL细胞分化过程中，我们检测到了几个基因的差异表达。此外，我们还将此cDNA微阵列应用于牙周组织基因表达的大规模分析。从牙周手术中采集的牙龈组织中提取总RNA，体外培养人牙龈成纤维细胞（HGF）。将RNA逆转录并用Cy 3和Cy 5差异标记，并杂交到微阵列上。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中检测到胶原和细胞粘附分子基因的上调。本研究结果表明，本研究建立的cDNA微阵列是一种分析牙周组织中大规模基因表达的有力工具，并提示利用该cDNA微阵列进行牙周疾病状况的多重分析的可能性

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