Effection Of Hypoxia On The Expression Of Matrixmetalloproteinases Of Human Periondontal Ligament Fibroblast Cells In Vitro

Objective:Periodontitis is a chronic inflammatory disease that is characterized by root square migration of junctional epithelium and progressive destruction of the tooth supporting apparatus. Although the pathogenesis of the various forms of this disease is not completely understood, Matrix metalloproteinases (MMPs), a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, are believed to have an important role in the pathological process of Periodontitis. Periodontitis, together with trauma and smoking, can cause local periodontal tissue hypoxia. Hypoxic environment has important biological effects on tissue and cells, however, it is not clear whether Hypoxia affects the expression of MMPs/TIMPs of human periondontal ligament fibroblast cells which is closely associated with the destruction of periodontium in periodontal diseases and thus plays an important role in the course of periodontal disease. To test this hypothesis, therefore, we exposure periodontal cells to hypoxia and normal oxygen tension (normoxia) to simulate the local ischemic model in periodontal tissue. The effects of hypoxia on the proliferation and mRNA expression of MMPs/TIMPs in human periodontal ligament cells were measured using Reverse transcription-polymerase chain reaction, which will has great significance on the better understanding of the pathological pathogenesis of periodontitis under anoxic condition and can provide an important basis for selecting the more accurate treatment method.Material and methods:1. The culture and identification of human periondontal ligament fibroblast cells Premolars extracted for orthodontic purposes were collected from health subjects from ten to twenty years old. Periodontal ligament tissue was removed from the mid-third of the root and minced with a surgical scalpel. All explants were placed into Dulbecco’s Modified Eagle’s Medium(DMEM), cultured and subcultured at confluence. Experiments were carried out with cells from the fifth passages. immunohistochemistry ABC metheod was performed to identify the source of cell.2. Experimental grouping In the control group, cells were incubated in a humidified atmosphere at normoxic conditions of20%O2,5%CO2and75%N2. In the hypoxia group, cells were incubated in a humidified atmosphere of1%O2,5%CO2and93%N2for12hours,24hours and48hours, respectively.3. The expression of MMP s and TIMPs in hPDLCs in vitro was measured using Reverse transcription-polymerase chain reaction. Cells were plated into6-well plate at an initial concentration of1×106cells/ml. According to experiment group, the cells were then incubated for12hours,24hours and48hours in different incubator, respectively. Subsequently, the total RNA was then extracted from each sample for Reverse transcription-polymerase chain reaction analysis. The expression of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, TIMP-2in hPDLCs in vitro was measured using Reverse transcription-polymerase chain reaction. The results were analyzed by SPSS13.0software package.Results:1. The expression of MMP-2, TIMP-1, TIMP-2mRNA in the hypoxic groups of12hours,24hours and48hours was statistically higher than that in the control groups while the expression of MMP-1mRNA under hypoxic conditions of48hours was slightly higher than that in the control groups.2. The expression of MMP-2mRNA in hypoxic groups showed a significantly increasing trend. There were significant differences between the hypoxic group and the normoxic control group about the expression of MMP-2mRNA in hPDLCs (p<0.01).3. The expression of MMP-1mRNA in hypoxic groups of12hours was momentarily decreased, and then increased with hypoxia time prolonged. There were no significant differences between the hypoxic group of12h and the normoxic control group about the expression of MMP-1mRNA in hPDLCs.4. The expression of TIMP-1, TIMP-2mRNA in hypoxic groups of12hours was momentarily increased; there were significant differences between the hypoxic12h group and the normoxic control group about the expression of TIMP-1, TIMP-2mRNA in hPDLCs (p<0.05). However, with hypoxia time prolonged, the expression of TIMP-1, TIMP-2mRNA in hypoxic groups showed a significantly declining trend and the expression changes between the hypoxic and the normoxic control group after hypoxic24h was not obvious.5. We do not detect MMP-9,13mRNA expression in the experiment and the result is not displayed.6. The change of ratio of MMP-1/TIMP-1mRNA in the hypoxic group of48h was not obvious compared with the normoxic group. The expression of MMP-1/TIMP-1mRNA in hypoxic groups of12hours was obviously decreased; there were significant differences between the hypoxic12h group and the normoxic control group about the expression of MMP-1/TIMP-1mRNA (p<0.05). The change of ratio of MMP-1/TIMP-1mRNA was obvious and showed a significantly rising trend in the hypoxia group.7. The ratio of MMP-2/TIMP-2mRNA in the hypoxia group significantly increased compared with normoxic group. There were significant differences between the hypoxic group and the normoxic control group about the expression of MMP-2/TIMP-2mRNA (p<0.05).Conclusion: Hypoxia could change the expression of MMPs/TIMPs mRNA and other relevant growth factors and also lead to the imbalance of MMP-2/TIMP-2mRNA expression. It is suggested that the imbalance of MMP-2/TIMP-2expression may be closely correlate with the occurrence and development of periodontal disease and play an important role in the process of periodontal tissue destruction in periodontitis.