| It has been wildly known that Reactive oxygen species (ROS) is involved in the pathological process of many diseases, and plays an important role in the normal metabolic reactions. ROS is involved in cancer, AIDS, atherosclerosis, chronic inflammatory state and aging process as well as a series of disease state. Oxidative phosphorylation of molecular oxygen as the terminal electron acceptor, occupies an important position in the aerobic metabolism, but this kind of electronic demand also led to the formation of various oxygen, such as 02-, OH and hydrogen extraction ability of H2O2, HC10 etc. Moreever, it can start a chain reaction, a variety of easy and cell membrane unsaturated fatty acids and cholesterol reaction, as the direct role of oxidative damage in cells can lead to cell apoptosis. But the organism in the process of evolution with anti ROS effective defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and antioxidants (such as vitamin C, vitamin E). Therefore, the metabolism of the body oxygen consumption at the same time the formation is often accompanied by ROS, and the state depends on the cells in ROS and the antioxidant capacity of balance.Periodontal disease is the highest incidence of infectious diseases in humans. As the initiating factor, bacteria is the main cause of adult tooth loss as the destructive disease. It has been believed that the oxidation/periodontal disease in saliva and gingival crevicular fluid of the antioxidant activity of the balance is broken, in which the periodontal membrane of these patients more susceptible to the effects of ROS damage. In this study it was found that the level of total antioxidant activity of saliva patients with periodontal disease compared with normal people do not change or drop. The imbalance of oxide/antioxidant levels and iron copper sources of change is to promote periodontal tissue damage, in the presence of ROS. Besides, periodontal ligament cells and gingival epithelial cells showed cell damage and cell lysis potential. SOD is mainly confined to periodontal membrane play a role, followed by collagen fibers and fibrolasts. With the increasing severity of periodontal disease, SOD and catalase activity decreased in gingival tissue.In this research, through clinical acquisition in healthy population of periodontal ligament tissue, periodontal ligament cells was cultured due to orthodontic patients aged 12 to 15 years old. The selected teeth are without caries periodontitis and periapical periodontitis. The teeth were immediately put into cell culture fluid transfer, and then joined by antibiotics in a super clean bench PBS repeated washing the root surface. Periodontal membrane and operation knife scraping root in 1/3, using cell culture fluid was washed two times with a pair of scissors, cut into pieces and then repeated the centrifuge tube, by enzyme digestion treatment, adding 0.1% collagenase 3ml digestion,37℃ oscillation of 50-60min, see the loose organization, with 1% trypsin to 0.125%, continue to digest 25min most of the cells, free, full blown by Straw disperse sufficiently, adding a small amount of 10%FBS cultured in PMI1640 termination of digestion. 1000rpm centrifugal 10min, Kami Kiyo, joined the 10%FBS PMI1640 medium allows cells to suspension, then inoculated in the culture bottle, add the appropriate amount of culture fluid, incubator in 37℃,95% air,5% CO2 and saturated humidity in. On next day the growth of cells was observed in 2 to 3 days, the medium was changed. At the same time, using tissue piece, with tissue shear is cut into 1mm* 1mm* 1mm about the size of small, inoculated in cell culture bottle, spread evenly, each distance about 5mm. When in place, the flask gently, adding proper amount of 10%FBS containing DMEM, into the CO2 incubator. After cultivation of 20-25 days, cells covered the bottom of the bottle about 90% from tissue pieces in the middle reaches eith 0.25% trypsin digestion passage. The fifth generation to the eighth generation cells for subsequent experiments. Through collection of log phase PDLC cells, the cell suspension concentration is adjust ed to inoculated in 96 well plate, to join 100u into each hole, the cell density to 1000-10000/hole to be measured, the edge hole with aseptic filling PBS.5%CO2, 37℃ to incubation, cell monolayer covered with the bottom of the hole, to be adherent cells completely in good condition, at a specific time (0h-48h) of drugs with different concentration of (VC) or compound (H2O2) treated cells, after staining with the PDLCs FACS with flow cytometry analysis. The PDLC cells were inoculated to 12 hole cell plates, with H2O2 and VC treatment of PDLCs for 3h and 6h after different doses of each hole with about 160ul of NP-40 cell lysate, the ice cracking cell 30min, repeated pipetting or the oscillator shocks on 5min, draw the cell lysate into pre cooling Ep tube,4 C 12000rpm centrifuge 10min, supernatant was transferred to another tube EP, used to detect the protein concentration of BCA kit. Results:MTT detection of different concentrations of H2O2 cell activity,600 M H2O2 treatment for 3 h, PDLCs activity was significantly decreased after. About 50% of the PDLCs death at 600 M after H2O2 treatment, and with the H2O2 concentration increased the number of deaths is relatively stable. In MTT it was found the different concentrations of H2O2 cell activity. Results show that 600 M H2O2 for 3 h, PDLCs activity was significantly decreased after. About 50% of the PDLCs death at 600 M after H2O2 treatment, and with the H2O2 concentration increased the number of deaths is relatively stable. At the same time to 6h the same concentration of H2O2 treatment time, similar results are also obtained. With a high concentration of 800 M and 1000 M cells treated with H2O2, the disappearance of normal cell shape, especially in the 1000 M concentration, cells became round. In the stimulation of 6h was observed after cell morphology was found the most obvious toxicity action. The study also found that with the increase of H2O2 concentration, the disappearance of shrinkage and complete structure of the cell phenomenon. In general, H2O2 time and dose dependently induced PDLCs cell death. With different doses of VC treatment for PDLCs, the cell viability of MTT method for detection of treatment, in addition to the results of VC treatment was the lowest concentration of 10 M, the other relatively high doses (from 50 to 200 mu M) VC will produce toxic effects on the cells without H2O2 treatment. Therefore, this research thinks, ROS has toxic effect on PDLC, H2O2 and caspase-9 induced apoptosis through the activation of the caspase-3 signaling pathway, the morphology and activity of PDLC, VC may promote apoptosis antagonizing the effect of H2O2, VC properly may be a potentially effective means of treatment of periodontitis.A number of studies confirmd that ROS plays a role in many pathological changes, including chronic periodontitis. Protein carbonylation are biomarkers of oxidative protein damage when the state, reflecting the injury of ROS cells induced by. The study on the protein of 13 periodontitis patients and 13 normal controls were detected in plasma carbonyl (PC) levels. Of all the subjects were examined and sampled detailed, protein carbonyl reagent kit for detecting the level of plasma. Results: compared with normal people, patients with chronic periodontitis peripheral blood protein carbonyl levels increased significantly (P<0.05). Detection of PC and PC levels in patients with concentration in plasma, the results showed that the PC level of chronic periodontitis group was 2.30 (0.94-3.17) nmol/mg, PC levels in healthy periodontal group was 1.42 (0.78-2.32) nmol/mg, chronic periodontitis group PC levels were significantly higher than that of periodontal healthy group (P<0.01). Chronic periodontitis group, the concentration of PC was 184.6 (84.6-269.4) nmol/ml, periodontal health group was 115.5 (49.3-165.3) nmol/ml, chronic periodontitis group PC concentration were significantly higher than those in periodontal healthy group (P<0.01) 0.592 is the coefficient of correlation of plasma PC level and periodontal parameters PD,0.673 is the coefficient of correlation of plasma PC level and CAL, and the coefficient of correlation of plasma PC concentration and periodontal parameters of PD is 0.657, and the correlation coefficient CAL is 0.55. Thus, it was believed there is significantly positive correlation between PD and CAL and the plasma PC level and the total. Conclusion:the higher level of protein carbonylation circulation system of chronic periodontitis patients, elevated PC levels may be a sign of oxidative damage of periodontitis. |
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