사람 피부 섬유아세포와 치은 섬유아세포에서 아라키돈산 처리에 따른 세포외기질 발현의 차이 Differential Expression of Extracellular Matrix-Associated Genes Between Dermal and Gingival Fibroblasts in Response to Arachidonic Acid
- 언어ENG
- URLhttp://db.koreascholar.com/Article/Detail/293141
pp.83-94
The pattern of wound healing process differs markedly according to the cell types. Gingival wounds heal more rapidly without scar, however dermal wounds show collagen laid down in thick disorganized patterns and keloid formation. This h as b een s uggested t o be d ue t o the presence of d ifferent E C M components a nd c ytokines a s well a s growth factors. The purpose of this study was to examine the differential expression of genes in connection with keloid formation in gingival fibroblasts (hGFs) and dermal fibroblasts (hDFs) in response to inflammation. In this study, we investigated the differences between hGFs and hDFs in the expression and production of cyclooxygenase (COX-2), prostaglandins E2 (PGE2), transforming growth factor (TGF)-β, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs) which play important roles in collagen deposition in wound healing. The hGFs and hDFs were primary cultured and allocated to arachidonic acid (AA) treatment group and control group. Protein and mRNA were extracted right after (0 hr) and 24 hr after AA treatment. At a defined concentration of AA in hGFs and hDFs, MTT assay was performed. The mRNA and protein expression levels of COX-2, TGF-β, collagen 1 and 3, MMP 1 and TIMP 1 were examined by Real-time PCR and Western blots. The amounts of PGE2 were measured by enzyme-linked immunosorbent assay (ELISA).The expression of COX-2 and TGF-β exhibited reduced levels in hGFs , but were increased in hDFs at 24 hr after AA treatment. Production of PGE2 was increased in hGFs and hDFs at right after AA treatment but, not changed at 24 hr after AA treatment. The protein and mRNA expression of collagen 1 and 3 were decreased in hGFs , whereas increased in hDFs at 24 hr AA treatment. Expression of MMP-1 protein was increased in hGFs at 24 hr but, was decreased in hDFs at 24 hr compared with that of control. The protein expression of TIMP-1 was decreased in hGFs but, was increased in hDFs at 24 hr compared with that of control. These observations demonstrate differential expression between gingival and dermal fibroblasts in regulation of collagenolytic capacity by extracellular matrix-associated genes in keloid formation associated with wound repair.
II. MATERIAL and METHOD
1. Cell Culture
2. Cell Treatment Condition
3. MTT Assay
4. RNA Isolation and Real - Time PCR
5. Western B lots Analysis
6. ELISA for Measuring PGE2 Levels in Media
III. Results
1. MTT Assay for Choose the AdequateConcentration of AA
2. The Expression of COX-2 and PGE2Activity After AA Treatment
3. The mRNA and Protein Expression ofTGF-β After AA Treatment
4. The mRNA and Protein Expression ofCollagen 1 and 3 After AA Treatment
5. The mRNA and Protein Expression ofMMP-1 After AA Treatment
6. The mRNA and Protein Expression ofTIMP-1 After AA Treatment
IV. DISCUSSION
V. CONCLUSION
VI. REFERENCES