| Background and objectivePeriodontitis is one of the most common oral diseases, about 80%-90% adults suffer from varying degrees of periodontal disease. Currently, periodontitis has become the first reason of tooth loss in audult. To comply with the classical theory, periodontal disease is an acute or chronic recurrent progressive inflammatory disease initiated by dental plaque and its products (such as lipopolysaccharide), which activate the host inflammatory response,and causes the periodontal support tissue inflammation or necrosis.Periodontal disease is closely related to periodontal bacteria inside tooth plaque, especially the gathering of Gram-negative (G-) anaerobic bacteria. Endotoxin as the lipopolysaccharide(LPS) in the outer membrane of the G- bacteria cell wall. LPS is one kind of highly reactive pathogenic substances in G- bacteria, which has very strong toxic and antigens effects on the periodontal tissue cells and plays an important role in the development of periodontal disease. LPS has different chemical composition in different microorganisms, but its basic structure is similar.LPS has three different composition with different structure and biological activities. They are lipid A,0- specific polysaccharide chain and core oligosaccharide. The organimmune active cells generate endogenous adjustment factors, forming a network of cytokines in the body. These cytokines are keys to the biological expression of LPS. In a certain sense,pathogenecity of LPS is reflected by its ability to stimulate the body to produce endogenous cytokines. To now,it is confirmed that the direct effect of bacteria on periodontal tissue damage is limited, and the host immune response stimulated by bacteria is the main cause of periodontal tissue destruction. Thus by inhibiting local immune response and blocking this process from producing inflammatory mediators can block the development of periodontal disease more effectively, so that periodontal disease can be controlled.Normally, periodontal tissue has the ability to regenerate and repair by itself mainly by human periodontal ligament cells. Periodontal ligament fibroblasts is the most important periodontal ligament cells, which has some biological characteristics, such as secreting and contracting collagen,forming mineralization,regenerating proliferation and adhesion.Also,it is the basis of periodontal tissue regeneration, restoration and attachment. During the occurrence and development of periodontal diseases, pathological necrosis or apoptosis of periodontal ligament cells eventually led to the loss of periodontal support and the loose teeth. Therefore, it is the key of periodontal therapy that there exist a mount of the periodontal ligament cells,and they can effectively adhere, proliferate and differentiate in root surface,and finally regenerate in periodontal tissue physiologically and functionally.Currently, it is an important adjunct method to cure the periodontal disease with medicine in clinic. sometimes with basic treatment to enhance and consolidate the effect. Broad-spectrum antibiotics and synthetic drugs have high efficient bacteriostasis and sterilization.But Large doses usage with a long time period is easy to produce adverse reactions such as dysbacteriosis and drug resistance,which limit the popular application in clinic. Traditional Chinese medicine as the quintessence of our country, can understand periodontal disease in a whole view, which not only focuses on the impact of gum disease risk factors, but also pays attention to the impact of periodontal host response,can compensate defect westernmedical treatment which emphase on local periodontal disease. This is also the advantage of traditional Chinese medicine in treating periodontal disease.Radix Hedysari, as the dried root of Hedysarum polybotrys belongingto an erect perennial herbaceous plant, has functions in consolidating superficies, replenishing blood, diuresis for detoxification,drainage and astringing wound for regenerating tissue. Hedysari radix polysaccharide (HPS), which is one of the main chemical constituents of Hedysari radix, composes rhamnose, xylose, arabinose,galactose and glucose. Recently,pharmacological research indicate that HPS possess the effects of immunomodulatory, antitumor and antioxidant. Furthermore. HPS may be considered as a new drug of traditional Chinese medicine with high value for development. This study was designed to investigate the proliferation of HPS on human periodontal ligament cells and the effect of HPS on the expression of IL-1β,IL-6 in human periodontal ligament cells treated with lipopolysaccharide (LPS), and explore the effect of HPS on human periodontal ligament cells.Chapter 1 The culture and identification in vitro of hPDLCsObjectiveCulture, identify and observe the features of hPDLCs’ shape in vitro.Methods1. PDLCs were obtained from periodontal health donors (age from 12-25) who underwent tooth extraction for orthodontic reasons. Cells were collected from the middle-third of the tooth roots under sterile conditions by tissue enzyme digestion method. Take primary and the third generation of cells, observe the growth and morphological characteristics under inverted phase contrast microscope.2. Pick up the 3rd generation cells with the growth in good condition, adjust the cell concentration to 5×104/mL. Before the cells were seeded in six-well plates,cover slips were placed. The cells were incubated at 37℃,5% CO2 incubator until cover slips were full. Remove the culture medium, wash the cells with PBS, the cover slips were fixed with 4% paraformaldehyde 20min. Observe vimentin and keratin staining under light microscope,take photos.3. Use MTT assay to detect the cytoactive of hPDLCs:Pick up the 3rd generation cells with the growth in good condition, the cells were seeded in 96-well plates at 1.0×104/hole.20μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at a concentration of 5 mg/mL was added to each culture well, at the indicated time points. After incubation at 37℃ for 4h, discarded the supernatant, and added 150μL dimethylsulfoxide to each well.The absorbance of each well was measured using a microculture plate reader with a test wavelength of 490 nm. Cell growth curve was drawn, the experiment was repeated three times.Results1. hPDLCs had higher grow rate and survival rate by the tissue enzyme digestion. After 5-10 days, the cells swam out from the surrounding tissue mass in primary culture. High magnification observation shows cells were spindle or star-shaped with plump body, uniform cytoplasm, central circular or oval nucleus and the visible nucleolus as fibroblast-like cells. The cells adhered within 24h after the cells were passaged. The passaged cells had fast growth rate, gradually stretched irregular circular, polygonal, spindle with radioactive proliferation.2. Immunohistochemical results showed that:vimentin staining was positive, cytokeratin negative, indicating that the cultured cells derived from mesenchymal tissue.3. hPDLCs growth experienced low growth of 1-2 days, rapid proliferation of 3 days, a plateau of 7 days.It charactered the law of fibroblast growth.Chapter 2 The proliferative activity of hPDLCs stimulatded with HPSObjectiveTo study the HPS on the proliferation of periodontal ligament cells, select the appropriate role of concentration as a foundation for subsequent experiments.Methods1. The culture of human periodontal ligament cells:the same as the chapter 1.2. The 4th generation hPDLCs were seeded in 96-well plates at 1.0 × 104/hole, cultured in 100μL DMEM containing 10% FBS,37℃、5% CO2 and saturated humidity conditions. Discarded the original culture medium when the cells were adherent, then added 0μg/mL, 0.01μg/mL, 0.1μg/mL, 1μg/mL, 10μg/mL, 100μg/mL of HPS solution, while without cells as the control group. In sample Id,3d,5d, the MTT assay was observed after treatment with different concentrations of HPS cell proliferation after periodontal ligament cells under normal culture. The effect of HPS on the proliferation of hPDLCs was measured with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay 1,3,5 d after treatment. the experiment was repeated three times.3. Statistical analysis:The statistical significance of data was evaluated using one way ANOVA and the SPSS 20.0 software, test level α=0.05. Test homogeneity of variance before analysis of variance. When the information consistent with homogeneity of variance, we used LSD method for multiple comparisons, on the contrary, used Dunnett T3 method for multiple comparisons.ResultsCompared with the control group, different concentrations of HPS had no promotion of hPDLCs proliferation (P> 0.05). At 3d and 5d, 0.01μg/mL,0.1 μg/mL, 1μg/mL, 10μg/mL can enhance the proliferation of hPDLCs. Group E (10μg/mL)has significant difference with the control group (P<0.05), high concentrations of 100μg/ mL (F group) reduced cell proliferation.Chapter 3 Effect of radix hedysari polysaccharide on expression of IL-1β, IL-6 in human periodontal ligament cells treated with LPSObjectiveTo observe the effect of Radix Hedysari polysaccharide(HPS) on the expression of IL-1β,IL-6 in human periodontal ligament cells (hPDLCs) treated with lipopolysaccharide (LPS) and to investigate the effect of HPS on the periodontal inflammatory reaction.Method1. The culture of human periodontal ligament cells:the same as the chapter 1.2. The 4th generation hPDLCs were seeded in 6-well plates at 1.0×106/mL, cultured in 100μL DMEM containing 10% FBS,37℃、5% CO2 and saturated humidity conditions. Discarded the original culture medium when the cells were adherent. Cells without stimulation served as the control and cells treated with LPS (10μg/mL) comprised the LPS group. The LPS +HPS groups were pretreated with HPS (10μg/mL) for 2h, and then treated with LPS. At 24 h, total RNA was extracted using TRIzol. Total RNA as a template in each group were reverse transcribed into cDNA, then IL-1β,IL-6 were examined by real-time quantitative polymerase chain reaction (qRT-PCR).3. The 4th generation hPDLCs were seeded in 24-well plates at 1.0×105/mL, cultured in 100μL DMEM containing 10% FBS,37℃.5% CO2 and saturated humidity conditions. Discarded the original culture medium when the cells were adherent. Cells without stimulation served as the control and cells treated with LPS (10μg/mL) comprised the LPS group. The LPS +HPS groups were pretreated with HPS (10μg/mL) for 2h, and then treated with LPS. at 24h, in accordance with the IL-1β, IL-6 ELISA kit’s instructions to calculate IL-1β, IL-6 in the cell medium.4. Statistical analysis:The statistical significance of data was evaluated using one way ANOVA and the SPSS 20.0 software, test level α= 0.05. Test homogeneity of variance before analysis of variance. When the information consistent with homogeneity of variance, we used LSD method for multiple comparisons, on the contrary, used Dunnett T3 method for multiple comparisons.Results1. qRT-PCR test results showed that IL-1β, IL-6 mRNA expression of periodontal ligament cells stimulated by 10μg/mL LPS were significantly increased, but after the addition of 10μg/mL of HPS, IL-1β and IL-6 mRNA were significantly reduced (P<0.01).2. ELISA test results are consistent with the qRT-PCR results. Stimulated by 10μg /mL LPS, the IL-1β, IL-6 in the culture medium were significantly increased, but after the addition of 10μg/mL of HPS, IL-1β, IL-6 were significantly reduced (P <0.01).Conclusions1. The cells cultivated with the tissue enzyme digestion method have high survival rate, and can still keep good cytoactive and amplication ability after many generarion of multiplication. According to the immunohistochemical results, the source of cells is reliable..2. A certain concentration of HPS can promote the proliferation of human periodontal ligament cells, while it has no effect at high concentrations.3. A certain concentration of HPS can inhibit the expression of IL-1β and IL-6 in human periodontal ligament cells treated with LPS, suggesting HPS can modulate LPS-induced inflammation by inhibiting IL-1β and IL-6 release in hPDLCs. |
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