Abstract
This study was undertaken to investigate the effect of oxygen tension on the activity and function of the cells derived from human periodontal ligament by measuring cell activity, total protein synthesis, collagen synthesis, $IL-1{\beta},\;IL-6,\;TNF-{\alpha}$ Human periodontal ligament fibroblasts were collected from premolars extracted for orthodontic treatment and incubated in the environment of $37^{\circ}C,\;5\%\;CO_2,\100\%$ humidity. After the fifth to sixth passage they were used for the experiment. Gaspack system to which $0.2{\mu}m$ Millipore filter was attached was connected to mixed-gas tanks. The mixed gases were composed of $10\%\;O_2,\;5\%\;CO_2,\;85\%\;N_2$ in hyoxic group or $90\%\;O_2,\;5\%\;CO_2,\;5\%\;N_2$ in hyperoxic group and $5\%\;CO_2,\;95\%$ air for control. After incubation in $37^{\circ}C$ for 2, 4, 6 days, cell activity was determined by tetrazolium(MTT) assay and total protein synthesis was assayed using sulforhodamine B(SRB). And measurement of 4-hydroxyproline was performed to assess collagen synthesis md $IL-1{\beta},\;IL-6,\;and\;TNF-{\alpha}$ were measured by enzymeimmunoassay. The results were as follows. 1. The cell activity and total protein synthesis in hypoxic group were a little higher than or almost the same with those in control group. 2. In hyperoxic group, the cell activity was lower than that in control group and total protein synthesis was decreased. 3. Collagen synthesis was significantly decreased initially in both hypoxic and hyperoxic group and increased nearly to the level of control group as the duration of cell incubation was longer 4. As a result of enzymeimmunoassay, the amount of cytokines was $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ in order. 5. $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ were increased more rapidly in both hypoxic and hyperoxic group than in control group as the duration of cell incubation was longer. 6. There were more $IL-6\;and\;TNF-{\alpha}$ in hyperoxic group than in control group after 6 days, and there were more $IL-6\;and\;TNF-{\alpha}$ after 6 days than after 2 or 4 days in hyperoxic group. These results suggested that oxygen tension might modulate the production of extracellular matrix and cytokines in the cells derived from human periodontal ligament.
본 연구는 산소 장력이 사람의 치주인대에서 유래된 세포의 활성과 세포외 기질 및 cytokine에 미치는 영향을 알아 보고자 저산소 및 과산소 조건에서 배양한 후 세포의 활성, 총 단백질 합성, 교원질 합성, 그리고 $IL-1{\beta}$, IL-6, $TNF-{\alpha}$를 측정하였다. 교정치료를 위해 발거한 제 1 소구치에서 사람의 치주인대 세포를 채취하여 $37^{\circ}C,\;5\%\;CO_2,\100\%$ 습도의 환경에서 배양한 후 5 내지 6 계대배양하여 실험에 사용하였다. Gaspack system에 $0.2{\mu}m$ Millipore filter를 부착하여 세균 감염을 막았고, 저산소군에는 $10\%\;O_2,\;5\%\;CO_2,\;85\%\;N_2$ 가스를, 과산소군에는 $90\%\;O_2,\;5\%\;CO_2,\;5\%\;N_2$ 가스를 연결하여 산소 장력에 변화를 주었으며 대조군에는 $5\%\;CO_2,\;95\%$ 공기를 공급하였다. $37^{\circ}C$ 에서 2, 4, 6일간 배양한 후에 세포활성을 측정하기 위해 tetrazolium(MTT) assay를 시행하였고 sulforhodamine B(SRB) assay를 이용하여 총 단백질 합성을 알아보았으며, 4-hydroxyproline 측정으로 교원질 합성을 측정해 보았다. 또한 효소면역측정법으로 $IL-1{\beta}$, IL-6, $TNF-{\alpha}$를 측정하여 다음과 같은 결과를 얻었다. 1. 저산소근에저 세포 활성과 총 단백질 합성은 대조군보다 다소 높거나 같은 정도이었다. 2. 과산소군에서 세포 활성은 대조군보다 낮았으며 총 단백질 합성도 다소 감소되었다. 3. 교원질 합성은 저산소군과 과산소군에서 대조군보다 유의하게 감소되었다가 배양기간이 길어짐에 따라 증가되어 대조군과 유사한 정도에 달했다. 4. 효소면역법 측정 결과 IL-6, $TNF-{\alpha},\;IL-1{\beta}$ 순으로 다량 검출되었다. 5. IL-6, $TNF-{\alpha},\;IL-1{\beta}$는 저산소군과 과산소군에서 배양기간이 길어짐에 따라 대조군에 비해 급격히 증가하였다. 5. IL-6, $TNF-{\alpha}$는 과산소 조건에서 배양 6일 후에는 대조군에 비해 유의하게 증가하였으며 이는 배양 2일 또는 4일 후보다도 유의하게 증가한 것이었다.