1998 Fiscal Year Final Research Report Summary
Application of in situ PCR to the diagnosis of oral diseases
| Project/Area Number |
08457562
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
矯正・小児・社会系歯学
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
OGUCHI Haruhisa School of Dent., Hokkaido Univ., Pro., 歯学部, 教授 (30124689)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIHARA Toshihiro School of Dent., Hokkaido Univ., Inst., 歯学部, 助手 (60261319)
KUDO Masaki School of Dent., Hokkaido Univ., Inst., 歯学部, 助手 (10164500)
SHIRAKAWA Tetsuo Dental Hospital, Hokkaido Univ., Lec., 歯学部, 講師 (00187527)
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| Project Period (FY) |
1996 – 1998
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| Keywords | herpes simplex virus / Mycoplasma salivarium / saliva / polymerase chain reaction / nitric oxide / nitric oxide synthase / in situ hybridization |
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| Research Abstract |
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.
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