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Purpose of the experiment by comparing the clinical collection of smoking and non-smoking chronic periodontitis gingival crevicular fluid (Gingival crevicular fluid, GCF) and healthy smokers and non-smokers gingival sulcus fluid matrix metalloproteinase-9 (Matrix metalloproteinase-9, MMP- 9) level of smoking impact of chronic periodontitis gingival crevicular fluid MMP-9, to infer and confirm the role of smoking on chronic periodontitis, provide a theoretical basis for the prevention and treatment of periodontal disease. The experiment also by the different concentrations of nicotine stimulation of cultured human gingival fibroblasts (Human gingival fibroblasts, HGFs), observed in the growth, proliferation activity in terms of the impact of different time periods with different concentrations of nicotine and a certain concentration of nicotine in vitro The HGFs secretion of MMP-9 culture, cell protein expression levels of MMP-9, and explore the effect of nicotine on the secretion of MMP-9 in gingival fibroblasts. Method 1 study of smoking and non-smoking healthy gingival sulcus fluid, smoking and non-smoking chronic periodontitis gingival crevicular fluid MMP-9 level. Acquisition in the clinical gingival sulcus fluid which MMP-9 level was measured by enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, ELISA). 2 (1) obtained in clinical normal gum tissue, using traditional tissue explant primary cultured human gingival fibroblasts, observation of cell morphology and cell immunohistochemical staining (2) the use of fourth-generation cells take growing well The fourth generation of cells into a single cell suspension of 1 × 104/ml, 100μl per well uniformly inoculated in 96 well plates with different concentrations of nicotine (10μg/ml, 50μg/ml, 100μg/ml, 250μg/ml, 500μg / ml, 1000μg/ml, 2000μg/ml) stimulate gingival fibroblasts, the use of the MTT colorimetric four (Methyl thiazolyl tetrazolium, MTT) to detect the relative growth rate of gingival fibroblasts in different concentrations of nicotine stimulation and growth inhibition rate, so as to observe the effect of nicotine on gingival fibroblasts in the growth and proliferation activity. (3) a cell density of 1 × 104 per well in 24-well plates, the fourth generation gingival fibroblasts inoculated on coverslips 24h adherent cells discarded after the original culture was washed with 2% DMEM culture medium resting 24h experimental group converter not Jianiguding stimulation using only 2% DMEM culture containing different concentrations of nicotine, 2% DMEM culture, control group, and then, respectively, in different time periods: Oh, 4h, 8h, 24h and 24 hours later, different concentrations: 1000μg / ml, 100μg/ml, Oμg / ml, prepared gingival fibroblasts cells crawl chip ice acetone fixed, the use of cellular immune staining method SP method to detect the amount of expression of MMP-9 in HGFs, which observed nicotine gums fibroblast secretion of matrix metalloproteinases. Results 1 patients with chronic periodontitis, smoking group, MMP-9 levels higher than the non-smoking group (P lt; 0.05). The chronic periodontitis smoking and non-smoking group, MMP-9 levels were significantly higher than the healthy group (P lt; 0.05). The healthy smokers MMP-9 level and non-smoking group no statistically significant difference. 2 different concentrations of nicotine stimulates gingival fibroblasts MTT results showed: compared with the negative control group, the first day of 10μg/ml group (A), 10μg/ml group (B), 100μg/ml group (C), 250μg/ml group (D), 500μg/ml group (E), 1000μg/ml group (F), (G) of HGF 2000μg/ml relative inhibition rate of 4.36%, 6.03%, 8.86%, 11.51%, 16.33%, 21.67 %, 25.26%; next day results: 9.45%, 14.66%, 18.11%, 22.76%, 25.65%, 34.12%, 39.52%; the third day of the results showed: 11.97%, 15.83%, 20.31%, 25.12%, 28.89%, 44.58%, and 50.66%, respectively. Three days seven inhibition rate was gradually increased and dose-dependent relationship, that is, the greater the concentration of nicotine, the higher the HGF inhibition rate. 3 Immunohistochemistry detected gingival fibroblasts in different time periods MMP-9 expression levels in nicotine stimulation results show: MMP-9 antibody mainly in the cytoplasm gingival fibroblasts, Oh, 4h, 8h, 24h , MMP-9 expression amount ± 0.0093,0.0774 ± 0.009, 0.0481 ± 0.0092,0.1296 ± 0.0225 .0980 (Oh, P lt; 0.01) 24 hours later, MMP-9 in the control group, 100μg/ml and 1000μg/ml expression were 0.0570 ± 0.0096,0.1296 ± 0.0225,0.2161 ± 0.0301 (compared with the control group, P lt; 0.01) expression trends gradually increased. Conclusions of the impact of smoking on chronic periodontitis GCF MMP-9 level changes. 2 nicotine can affect the proliferative activity of gingival fibroblasts: nicotine inhibit the proliferation of its toxic effects, and with the prolonged inhibition rate is getting higher and higher. Gingival fibroblasts small amount of MMP-9 expression in normal circumstances, a significant increase in the amount of matrix metalloproteinase-9 expression by nicotine stimulation.
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