Kim David M, Ramoni Marco F, Nevins Myron, Fiorellini Joseph P
Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA 02115, USA.
J Periodontol. 2006 Jun;77(6):1043-50. doi: 10.1902/jop.2006.050254.
There are no specific bacterial profiles or diagnostic tests capable of identifying refractory periodontitis patients before a treatment regimen is initiated. Therefore, in this high-risk cohort of patients who do not respond appropriately, host factors that might be partly under genetic control may play a crucial role in their susceptibility. Specifically, we tested the hypothesis that patients with refractory periodontitis have multiple upregulated and/or downregulated genes that might be important in influencing clinical risk.
Oral subepithelial connective tissues were harvested aseptically from seven refractory periodontitis and seven periodontally well-maintained patients. An RNA isolation kit was used to isolate total RNA from tissue samples that had been stabilized in the RNA stabilizing reagent. The isolated total RNA was then subjected to gene expression profiling using the microarray to measure gene expression levels. The retrieved data were analyzed with a computer program for the differential analysis of gene expression microarray experiments. In addition, real-time polymerase chain reaction (PCR) analysis was performed on selected samples to confirm the microarray data's gene expression patterns.
A total of 68 upregulated and six downregulated genes were identified that were differentially expressed at least two-fold out of 22,283 genes we analyzed. The selected model provided a 93% intrinsic validation along with a 93% extrinsic validation. To validate the microarray data, five upregulated genes (lactotransferrin [LTF], matrix metalloproteinase-1 [MMP-1], MMP-3, interferon induced-15 [IFI-15], and Homo sapiens hypothetical protein MGC5566) and two downregulated genes (keratin 2A [KRT2A] and desmocollin-1 [DSC-1]) were randomly selected for further analysis by real-time PCR. The relative RNA expression level of these genes measured by real-time PCR was similar to those measured by microarrays.
The combined use of microarray technology with the computer program for the differential analysis of gene expression microarray experiments provided a set of candidate genes that may serve as novel therapeutic intervention points and improved diagnostic and screening procedures for high-risk individuals.
在开始治疗方案之前，没有特定的细菌谱或诊断测试能够识别难治性牙周炎患者。因此，在这群反应不佳的高危患者中，可能部分受基因控制的宿主因素在其易感性中可能起关键作用。具体而言，我们检验了这样一个假设，即难治性牙周炎患者有多个上调和/或下调的基因，这些基因可能对影响临床风险很重要。
从7名难治性牙周炎患者和7名牙周状况良好的患者中无菌采集口腔上皮下结缔组织。使用RNA分离试剂盒从已在RNA稳定试剂中稳定保存的组织样本中分离总RNA。然后使用微阵列对分离的总RNA进行基因表达谱分析，以测量基因表达水平。检索到的数据使用计算机程序进行分析，以进行基因表达微阵列实验的差异分析。此外，对选定的样本进行实时聚合酶链反应（PCR）分析，以确认微阵列数据的基因表达模式。
在我们分析的22283个基因中，共鉴定出68个上调基因和6个下调基因，其差异表达至少为两倍。所选模型的内部验证率为93%，外部验证率也为93%。为了验证微阵列数据，随机选择了5个上调基因（乳铁传递蛋白[LTF]、基质金属蛋白酶-1[MMP-1]、MMP-3、干扰素诱导蛋白15[IFI-15]和人类假定蛋白MGC5566）和2个下调基因（角蛋白2A[KRT2A]和桥粒芯胶蛋白-1[DSC-1]）通过实时PCR进行进一步分析。通过实时PCR测量的这些基因的相对RNA表达水平与通过微阵列测量的水平相似。
将微阵列技术与用于基因表达微阵列实验差异分析的计算机程序结合使用，提供了一组候选基因，这些基因可能作为新的治疗干预点，并改善高危个体的诊断和筛查程序。
