사람의 치수, 치은, 치주인대 세포에 tumor necrosis factor (TNF)-α로 자극 시 matrix metalloproteinase (MMPs)의 분비에 관한 연구
The effect of tumor necrosis factor (TNF)-αto induce matrix metalloproteinase (MMPs) from the human dental pulp, gingival, and periodontal ligament cells
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연구목적: 본 연구는 in vitro 상에서 치수, 치은, 치주인대 세포를 neuropeptide (substance P, Calcitonin gene related peptides (CGRP))및 inflammatory cytokine (TNF-α) 으로 자극 시 matrix metalloproteinase (MMPs)의 생성 및 발현을 관찰한 것으로 치아와 치아 주변 조직에 염증이 존재할 경우 neuropeptide 및 inflammatory cytokine과 치아 경조직혹은 치조골의 remodeling에 중요한 역할을 하는 matrix metalloproteinase (MMPs)와의 관계를 규명하고자 하였다. 연구 재료 및 방법: 시편으로는 우식이 없는 건전한 제3대구치(n = 10)를 사용하였으며, 발거 후 즉시 Phosphate buffered saline (PBS)에 보관하고 치아에서 치은과 치주인대 조직을 채취하였다. 치아를 종축으로 절단하고 치수 조직을 채취하여조각으로 분리한 후 시편을 PBS에서 세 번 세척하였다. Plate에 치수, 치은, 치주인대 시편 조각을 위치시켜 Dulbecco’s Modified Eagle Medium (DMEM)을 첨가하여 세포를 배양하였다. 치수, 치은, 치주인대 세포를 culture dish에서 confluence에도달할 때 까지 배양하여 Fetal Bovine Serum (FBS)가 포함되지 않은 배지로 교환하여, 37℃에서 24시간 동안배양한 후 Phosphate buffered saline (PBS)로 1회 세척하고 Substance P (10-8 M, 10-5 M)가 포함된 배양액과 Mock (배양액만 포함됨)으로 4시간, 24시간동안 자극하였다. CGRP (10-6 M)을 함유한 배양액 및 TNF-α(2 ng/mL)를 포함한배양액으로 각각 24시간동안 세포를 자극하였다. 각각 다른 농도의 TNF-α(2 ng/mL, 10 ng/mL, 100 ng/mL)를 포함한배양액으로 24시간 동안 세포를 자극 한 후 RNase protection assay 및 Enzyme linked immunosorbent assay를 시행하였다. 결과: SP와 CGRP는 치수, 치은, 치주인대 세포의 MMPs발현에 관여 하지 않았다. TNF-α로 24시간 자극 시 치수, 치은,치주인대 세포에서 MMP-1,-12, -13의 발현을 증가시켰다. 반면, TNF-α는 치수, 치은, 치주인대 세포들에서 TIMP-3의발현을 감소시켰다. 서로 다른 농도의 TNF-α(2 ng/mL, 10 ng/mL, 100 ng/mL)로 24시간 자극 시 MMP-1과 MMP-13의 발현이 증가하였다. 결론: 치아와 치아 주변 조직에 염증이 존재 시 TNF-α가 증가함에 따라 치수, 치은, 치주인대로부터의 치아의 경조직 혹은치조골의 remodeling에 관여하는 matrix metalloproteinase (MMPs)가 중요한 역할을 하는 것으로 사료된다.
Abstract
Objectives: In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-α. Materials and Methods: The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-α, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay. Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP (10-5, 10-8 M) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP (10-5 M) and TNF-α(2 ng/mL) for 24 hrs and with various concentraion of TNF-α(2,10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8,7, 8, 9, 12, and TIMP 2, 3. In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-α(2,10, and 100 ng/mL) for 24 hrs and with TNF-α(10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13. Results: The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-αwere upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-αwere downregulated. TNF-α(2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs. Conclusions: TNF-αin the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone. [J Kor Acad Cons Dent 2011;36(1):26-36.]
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