| Objective：①To establish the model of human gingival fibroblasts（HGFs）subjected to compression force，and explore the biological effects of mechanicalstress on HGFs.②T o investigate the effects of hirudin on COL-Ⅰand MMP-1expression in HGFs followed by stimulation with compression force, andexplore the role and mechanism of hirudin on orthodontic gingival remodeling.Methods:①H GFswas obtained from explants of human normal gingivaltissues with tissue-explant method. Inverted phase-contrast microscopewas used to observe the cell growth situation. The3passage of HGFs wasidentified by immunochemistry of vimentin and cytokemtin.②The3-6passageof HGFs was plated onto6-well plastic plates at density of1×105/ml. After thecells grown to confluence，they were subjected to0,1,2,3,4,5g/cm2ofcompression force for24hrs,the compression of0g/cm2group was designatedthe control group. The MTT assay was performed to assess the changes in cellproliferative activity after application of compression force.③The experimentswere carried out at passage3-6. After HGFs were subjected to0,1,2,3,4g/cm2ofcompression force for24h，the mRNA expression of COL-Ⅰand MMP-1of HGFs were investigated through RT-qPCR，and the protein level of COL-Ⅰand MMP-1were detected by ELISA method.④The3-6passage of HGFs wasplated onto6-well plastic plates. After the cells grown to confluence，they weretreated with different concentrations of hirudin（0,0.5,1,2,4u/ml）and followed bystimulation with compression force（3g/cm2）for24h.The concentration of0u/mlhirudin group was used as control.The mRNA levels of COL-Ⅰand MMP-1were investigated through RT-qPCR，and the protein levels of COL-ⅠandMMP-1was detected by ELISA.Results：①Fibroblast-like cells were found growing around the gingival tissueexplants1-2weeks after initial incubation. The plasma-dominant staining forvimentin, brown granules in the cytoplasm and a negative staining forcytokeratin confirmed the mesenchymal origin of the cells.②The result of MTTshowed as follows：The OD value of all the groups was higher than controlgroup except the compression of5g/cm2group（P＜0.05）. The peak of ODvalue after application of the compression force was observed at3g/cm2.③Theresults of RT-qPCR and ELISA were consistent. Comparing with control group，treatment of HGFs with application of compression force for24h significantlyincreased the mRNA and protein expression of COL-Ⅰ.The peak of COL-Ⅰexpression after application of the compression force was observed at3g/cm2.④Treatment of HGFs with application of compression force for24hsignificantly decreased the mRNA and protein expression of MMP-1in allgroups except the compression of1g/cm2group（P＜0.05）.⑤Hirudinsignificantly down-regulated the expression of COL-I and up-regulated theexpression of MMP-1in HGFs induced by compression force stimulation. Theexpression of COL-I was increased and the expression of MMP-1was decreased in dose-dependent manner by hirudin.Conclusion:①Compression force could promote cell proliferation after HGFswere subjected to1,2,3,4g/cm2of compression force for24h.The compression of3g/cm2was employed as optimal force，since the peak of cell proliferation afterHGFs were applicated to compression force was observed at3g/cm2.②Compression force could elicit change in the metabolism of ECM byup-regulating the expression of COL-I and down-regulating the expression ofMMP-1in HGFs.③Hirudin could inhibit the deposition of collagen andpromote the degradation of collagen by up-regulating the expression of MMP-1and down-regulating the expression of COL-I，thereby facilitating gingivalremodeling during orthodontic tooth movement. |
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