Effects of cytokines on cell viability in Porphyromonas gingivalis-infected KB cells = Porphyromonas gingivalis에 감염된 KB 세포의 손상에 대한 cytokine의 효과

국문 초록 (Abstract)

치주질환에 있어서 Porphyromonas gingivalis는 질환을 일으키는 매우 중요한 요소이다. 이 연구의 목적은 P. gingivalis에 감염된 구강상피세포의 손상에 대한 cytokine의 효과를 살피는 것이다. 이번 연구에서는 P. gingivalis 초기감염에서 cytokine의 효과를 살펴보기 위해 적은 수의 P. gingivalis (MOI=10)가 사용되었으며, cytokine (IL-1β, TNF-α, INF-γ) 단독처리, P. gingivalis 단독처리, cytokine과 P. gingivalis 병용처리에 따른 세포손상, 세포주기, ROS (H₂O₂, O₂^(·-)) 생성과 NADPH oxidase mRNA의 발현의 변화를 평가하였다.
KB세포에 cytokine (IL-1β, TNF-α, INF-γ) 단독처리와 P. gingivalis (MOI=10) 단독처리시에는 세포손상이 미미하였으나 병용처리시에는 12시간부터 유의한 세포손상이 관찰되며 (≥30%), 세포의 손상 심화정도는 P. gingivalis와 TNF-α, P. gingivalis와 INF-γ, P. gingivalis와 IL-1β 병용처리 순서였다. 세포주기 변화는 cytokine 단독처리시 G0/G1기의 세포수 증가 경향을 보였고, cytokine과 P. gingivalis 병용처리시에는 S기의 세포수 증가 경향이 관찰되었다.
또한 KB세포의 ROS 생성은 병용처리시에 단독처리군보다 더 많은 H₂O₂ 생성을 보였으나 O₂^(·-) 생성은 모든 경우에서 변화가 관찰되지 않았다. NADPH oxidase mRNA 발현의 변화도 cytokine 단독처리시 NOX4와 Rac2가 24시간에 증가하였고, 병용처리시에는 24시간에 Rac2, p22^(phox)는 증가하였으나 NOX4와 NOX2는 감소하는 경향을 나타내었다.
이상의 결과를 요약하면 P. gingivalis에 감염된 KB 세포에 cytokine 처리는 세포손상을 현저히 심화시켰으며 세포손상기전에는 H₂O₂와 같은 활성산소종의 생성 증가에 기인할 수 있음을 시사하였다. 따라서, P. gingivalis에 감염시 cytokine의 존재가 치주질환의 형성과 진행과정y에 중요한 역할을 하리라 사료된다.

다국어 초록 (Multilingual Abstract)

The Porphyromonas gingivalis has been implicated as an important bacterial species in pathogenesis of periodontitis. The purpose of this study was to investigate the effects of cytokines on cell viability in P. gingivalis-infected KB cells. In this study, low numbers of P. gingivalis (MOI=10) was used to see the effects of cytokines in the early phase of P. gingivalis infection. The cell viability, cell cycle, intracellular reactive oxygen species (ROS) generation, and NADPH oxidase mRNA expression level were assessed in response to treatment of cytokine (IL-1β, TNF-α, or INF-γ), treatment of P. gingivalis, or combination treatment of cytokine and P. gingivalis.
When KB cells treated with cytokine or P. gingivalis alone, cell viability was slightly decreased (□10%). On the other hand, when KB cells were treated with combination of cytokine and P. gingivalis, cell viability was profoundly decreased at 12hr incubation (≥30%). The degree of severity in cell death was in the order of ①P. gingivalis - IL-1β, ②P. gingivalis - INF-γ, ③P. gingivalis - TNF-α combination treatment.
The results of cell cycle analysis by flow cytometry showed the inhibition of cell growth at G0/G1-phase in cytokine-treated groups and S-phase in combination-treated groups.
Intracellular ROS production of KB cells showed higher levels of H₂O₂ in combination-treated groups than in cytokine-treated or P. gingivalis-treated groups. However, the levels of superoxide anion was not changed in any condition. The expression level of NADPH oxidase mRNA showed increased NOX4 and Rac2 expression at 24 hr in cytokine-treated group. In combination-treated groups, increased expression of Rac2 and p22^(phox) and decreased expression of NOX4 and NOX2 were observed at 24hr.
In summary, the cytokine treatment resulted in severe cell death in P. gingivalis-infected KB cells. And increased H₂O₂ production might be involved in cell death. Therefore, cytokine seems to play an important role in early phase of P. gingivalis infection of periodontitis.

목차 (Table of Contents)

• Abstract = 1
• Ⅰ. Introduction = 3
• Ⅱ. Materials and Methods = 5
• Ⅲ. Results = 11
• Ⅳ. Discussion = 23
• References = 29
• 국문초록 = 36