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)的聚合酶链式反应(PCR)检测方法，并对这两种病原体在唾液中的流行情况进行了评价。快速聚合酶链式反应检测结果显示，241例健康儿童唾液标本中有17.0%呈HSV阳性，且无明显症状。来自健康志愿者(年龄：5-25岁)的唾液样本中有一半被发现为MS阳性。对于切片中DNA片段的原位检测，原位杂交和生物素-酪胺信号放大比传统的原位PCR方法更有利于DNA的显示。我们重点研究了一氧化氮(NO)作为炎症的生物标志物，据报道它存在于包括口腔组织在内的许多器官中。体外培养的人牙周膜细胞产生NO，用周期性张力力刺激细胞产生NO。在未经刺激的PDL细胞培养中，在新更换的培养液中，NO^2-和NO^3-(NO^2-/NO^3-)的浓度在最初的12h内增加到初始值的140%。反之，当细胞受到循环张力作用12h时，NO2-/NO3-增加3倍。我们用RT-PCR方法检测了PDL细胞中NO合成酶的mRNA。内皮型一氧化氮合酶mRNA在刺激和未刺激的牙周膜细胞中均有表达，而诱导型一氧化氮合酶mRNA在两种培养条件下均未检测到。这些结果提示：1)用聚合酶链式反应检测致病微生物或炎症标志物可用于口腔感染性疾病的诊断；2)人牙周膜细胞通过ecNOS产生一氧化氮，机械刺激的牙周膜细胞通过上调一氧化氮的产生来调节牙周组织的功能。

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.