Application of in situ PCR to the diagnosis of oral diseases

建立了单纯疱疹病毒（HSV）和唾液支原体（MS）的聚合酶链反应（PCR）检测方法，并评价了这两种病原生物在人唾液中的流行情况。快速PCR检测显示17.0%的健康儿童（n=241）唾液样本呈hsv阳性，无症状表现。健康志愿者（年龄：5-25岁）的唾液样本中有一半呈ms阳性。对于原位切片DNA片段的检测，与传统的原位PCR方法相比，原位杂交和生物素-酰胺信号扩增更有利于DNA的成功可视化。我们重点研究了一氧化氮（NO）作为炎症的生物标志物，据报道，一氧化氮在包括口腔组织在内的许多器官中都会产生。培养的人牙周韧带（PDL）细胞产生一氧化氮，并通过循环张力刺激细胞增强一氧化氮的产生。在未受刺激的PDL细胞培养中，在新交换的培养基中，NO^2-和NO^3-的浓度（NO^2-/NO^3-）在前12小时内增加到初始值的140%。相比之下，当细胞受到循环张力作用12 h时，NO2-/NO3-的含量增加了3倍。我们采用RT-PCR方法检测了PDL细胞中NO合成酶mRNA的含量。内皮NO合成酶mRNA在受刺激和未受刺激的PDL细胞中均有表达，而诱导型NO合成酶mRNA在两种培养条件下均未检测到。以上结果提示：1)PCR检测病原菌或炎症标志物对口腔感染性疾病的诊断具有重要意义；2)人PDL细胞通过ecNOS产生NO，机械刺激PDL细胞通过上调NO的产生调节牙周组织功能。

A polymerase chain reaction (PCR) assay for the detection of the herpes simplex virus (HSV) and Mycoplasma salivarium (MS) was developed and the prevalence of those pathogenic organisms in human saliva was evaluated. The rapid PCR assay revealed HSV-positive saliva samples in 17.0% of the total samples from healthy children (n=241) with no symptomatic manifestation. A half of the saliva samples from healthy volunteers (age : 5-25) was revealed to be MS-positive. For the detection of the DNA fragment in sections in situ, in situ hybridization followed by the signal amplification with Biotin-Tyramide was more advantageous for the successful visualization of the DNA than conventional in situ PCR method.We focused on nitric oxide (NO) as a biological marker of an inflammation, which is reported to be produced in many organs including oral tissues. Cultured human periodontal ligament (PDL) cells produced NO and the production was enhanced by stimulating the cells with cyclic tension forces. In unstimulated PDL cell culture, concentration of NO^2- and NO^3- (NO^2-/NO^3-) increased to 140% of the initial value during the first 12 h in newly exchanged medium. In contrast, NO2-/NO3- showed a 3-fold increase when the cells had been subjected to cyclic tension forces for 12 h. We employed RT-PCR method for the detection of NO synthases mRNA in the PDL cells. Endothelial NO synthases mRNA was expressed in both stimulated and unstimulated PDL cells whereas inducible NO synthases mRNA was detected in neither culturing condition.These results suggest that 1) detection of the pathogenic organisms or the marker of inflammation by PCR is useful for the diagnosis of infectious oral diseases, and 2) human PDL cells produce NO by ecNOS and mechanically stimulated PDL cells modulate the functions of periodontal tissue by the upregulated NO production.