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Objective:To detect the mRNA expression levels of inflammatory-related factors NLRP3, caspase-1, IL-1β, and IL-18in the murine macrophages infected by periodontitis patient's own tissue nucleic acid, and to discuss the effects of periodontitis patient's own tissue nucleic acid on the inflammation-related factors in the macrophages.Methods:The inflammatory periodontal tissue samples were collected during periodontal flap surgery of the chronic periodontitis patients, and the healthy periodontal tissue samples were collected from the patients without any periodontal diseases undergoing crown lengthening surgery.Then the total RNA from gingival tissue was extracted and reversely transcribed into cDNA.The cultured mouse macrophages RAW264.7were divided into control group and experiment group, then the healthy periodontal tissue cDNA and inflammatory periodontal tissue cDNA (the cDNA at a concentration of 1mg·L-1) were added into the RAW264.7cells, respectively.Real-time PCR was used to detect the mRNA expression levels of NLRP3, Caspase-1, IL-1β, and IL-18in the macrophages in various groups at 4, 6and 8hafter incubation.Results:The microscope observation showed that the mouse macrophages RAW264.7grew well with round and polygon shapes, clear cytoplasm, and full cell body.Compared with control group, the expression levels of NLRP3, Caspase-1, IL-1β, and IL-18mRNA in the RAW264.7cells in experiment group at 4, 6, and 8hwere increased significantly (P＜0.05or P＜0.01) , and the expression levels of NLRP3, Caspase-1, IL-1β, and IL-18 mRNA in RAW264.7 cells at 6 hin experiment group were the highest.Conclusion:The periodontitis patient's own tissue nucleic acids can promote the mRNA expressions of inflammation-related factors in the RAW264.7cells, suggesting that the periodontitis patient's own tissue nucleic acid has an immunomodulatory effect on the activation of RAW264.7cells.
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Objective:To investigate the effect of MT01 on the differentiation of osteoblasts under infected condition through determining the expression level of collagen Ⅰ (Col Ⅰ )mRNA in MG63 cells treated with Porphyromonas gingivalis (Pg).Methods:The cultured MG63 cells were divided into blank control,MT01,Pg, and MT01+Pg groups.MT01 at a concentration of 1 mg·L-1 was added into the MG63 cells,and the cells were incubated for 3 h.The cells treated with PBS (1 mg·L-1 )were used as control group.Then Pg (MOI=100∶1) was added.Real-time PCR was used to detect the expression levels of ColⅠ mRNA in MG63 cells at 2,4,6,8, 12 and 24 h after incubation.Results:Compared with blank control group,the levles of ColⅠ mRNA in the MG63 cells in MT01 and MT01 + Pg groups had no significant changes at 2 and 4 h (P > 0.05);the Col Ⅰ mRNA expression levels in MT01 group at 8,12 and 24 h were increased (P < 0.05 or P < 0.01).Compared with Pg group,the expression levels of ColⅠ mRNA in MT01+ Pg at 2 and 4 h were decreased,but the expression levels of ColⅠ mRNA were increased at 6,8,12 and 24 h (P <0.05 or P <0.01).Conclusion:MT01 can up-regulate the expression level of Col Ⅰ mRNA in the infected MG63 cells; MT01 could promot the differentiation of osteoblasts under infected condition.
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<p><b>OBJECTIVE</b>This paper aimed to determine the mRNA expression of osteoclast-related factors interleukin-6 (IL-6), interleukin-12 (IL-12) p35, IL-12p40, matrix metalloproteinase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATcl), receptor activator of nuclear factor-KB (RANK), and tumor necrosis factor-α (TNF-α) mRNA in murine macrophages infected by a periodontitis patient's own tissue nucleic acid. Another aim was to investigate the effects of a periodontitis patient's own tissue nucleic acid on the differentiation of macrophages into osteoclasts.</p><p><b>METHODS</b>Inflammatory periodontal tissue samples of chronic periodontitis patients were taken during periodontal flap surgery, and healthy gingival tissue samples were taken from orthodontic patients during tooth extractions. Total RNA from periodontal tissue was extracted and reversely transcribed into cDNA and then cryo-preserved until further use. First, specific sequence oligodeoxynucleotide MT0I at a concentration of 1 µg · mL⁻¹ was added in murine macrophage RAW264.7, and the cells were incubated for 3 hours. Cells with PBS (1 µg · mL⁻¹) were used as negative controls. The inflammatory periodontal tissue cDNA and healthy periodontal tissue cDNA (1 µg · mL⁻¹) was added subsequently. There were four experimental groups: healthy periodontal tissue cDNA+ RAW264.7, inflammatory periodontal tissue cDNA+RAW264.7, MT01+healthy periodontal tissue cDNA+RAW264.7, and MT01+inflammatory periodontal tissue cDNA+RAW264.7. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of osteoclast-related factors IL-6, IL-12p35, IL-12p4O, MMP-9, NFATcl, RANK, and TNF-α mRNA after 3, 6, 12, and 24-hours.</p><p><b>RESULTS</b>The mRNA levels of osteoclast-related factors NFATc1, MMP-9, TNF-a, IL-6, IL-12p40, IL-12p35, and RANK in RAW264.7 were markedly upregulated with the treatment of periodontitis patient's own tissue nucleic acid. However, the mRNA expression of osteoclast-related factors was inhibited by use of an immunosuppressant MT01.</p><p><b>CONCLUSION</b>The periodontitis patient's own tissue nucleic acid could promote the differentiation of murine macrophage into osteoclasts.</p>
Subject(s)
Animals , Humans , Mice , Cell Differentiation , Cytokines , Metabolism , Gene Expression , Gingiva , Interleukin-12 Subunit p40 , Interleukin-6 , Macrophages , Matrix Metalloproteinase 9 , Osteoclasts , Metabolism , Periodontitis , RNA, Messenger , Tumor Necrosis Factor-alphaABSTRACT
<p><b>OBJECTIVE</b>This aimed to investigate the effect of specific sequence oligodeoxynucleotide MT01 on the biological properties of osteoblasts invaded by Porphyromonas gingivalis (P. gingivalis ) by evaluating proliferation, cell cycle, and apoptosis.</p><p><b>METHODS</b>MG63 osteoblasts were recovered and incubated with MT01, CpG ODN, metronidazole (MNZ), and gentamicin (GEN) for 3 h. P. gingivalis (the multiplicity of infection was 100:1) was added subsequently and cocultured for another 24 and 48 h. Cells with PBS comprised the blank group, whereas cells with P. gingivalis comprised the negative controls. Six experimental groups were established: PBS group, P. gingivalis group, MT01+P. gingivalis group, CpG ODN+ P. gingivalis group, MNZ+P. gingivalis group, and GEN+P. gingivalis group. The proliferative ability was measured by methyl thiazolyl tetrazolium assay, and the percentages of apoptosis and cell cycle were examined by flow cytometry.</p><p><b>RESULTS</b>Compared with the blank group, proliferation increased significantly in the MT01+P. gingivalis group (P < 0.05). The ratio of cells was lower at the G₁ phase and higher at the S phase in the MT01+P. gingivalis group compared with the results in the P. gingivalis group (P < 0.05). Early cell apoptosis in the MT01+P. gingivalis group was significantly lower than that in the P. gingivalis group (P < 0.05).</p><p><b>CONCLUSION</b>MT01 can promote the proliferation, reduce the ratio of the G₁phase, increase the ratio of the S phase, and inhibit the early apoptosis of osteoblasts invaded by P. gingivalis.</p>
Subject(s)
Humans , Apoptosis , Cell Cycle , Cell Division , Cell Proliferation , Flow Cytometry , Gentamicins , Pharmacology , Metronidazole , Pharmacology , Oligodeoxyribonucleotides , Pharmacology , Osteoblasts , Cell Biology , Porphyromonas gingivalis , VirulenceABSTRACT
<p><b>OBJECTIVE</b>To study the effect of basic fibroblast growth factor (bFGF) on the gene expression of syndecan-4 by human periodontal ligament cell (PDLC) in culture, and discuss the effect of bFGF on human PDLC proliferation and migration.</p><p><b>METHODS</b>68 adolescent (12-18 years old) health premolar were collected, which were extracted for orthodontic reason. Human PDLC were cultured and stimulated by exogenous bFGF. After cultured 24, 48, 72h, gene expression of syndecan-4 was detected by SYBR green quantitative real time polymerase chain reaction.</p><p><b>RESULTS</b>The mRNA expression of syndecan-4 in 24 h group increased markedly than that in control group (P < 0.01), expecially in 1.0 ng x mL(-1) group. 1.0 ng x mL(-1) group in 48 h higher than that control group (P < 0.05). 1.0 ng x mL(-1) group in 72h compared with control group was lower (P < 0.05).</p><p><b>CONCLUSION</b>The mRNA expression of syndecan-4 was increased by bFGF at the beginning, but the expression was decreased with the time. The expression of such changes may be one of the important factors which participate in the migration process of PDLC.</p>
Subject(s)
Humans , Cells, Cultured , Fibroblast Growth Factor 2 , Periodontal Ligament , RNA, Messenger , Syndecan-4ABSTRACT
Objective To explore the effect of collagen membrane combined with recombinant human bone morphogenetic protein-2(rhBMP-2) on periodontal tissue regeneration and repair in periodontal defect models of rats.Methods Eighteen male healthy Wistar rats,which were made experimental periodontal bone defects in the inferior incisors,were randomly divided into three groups:control group,collagen membrane group(Co),collagen membrane and rhBMP-2 group(Co/rhBMP-2).Two rats from each group were sacrificed at 4,6,8 weeks after operation.The mandibles were removed,and examined under light microscope.Results In Co/rhBMP-2 group: 4 weeks after operation,a large amount of bone formed in the defects;6 weeks later,new bone filled in the defects;8 weeks later,alveolar bone emerged,newly-formed periodontal ligament and cementum localized at the root edges,no gingival epithelium was observed.In Co group:4 weeks after operation,new bone formed at the edge of the defects;6 weeks later,a larger amount of bone and periost were observed;8 weeks later,a small amount of periodontal ligament and cementum localized at the root edges.In control group: 4 weeks after operation,there was a small amount of newly-formed bone at the edge of the defects;6 weeks later,collagen fibers were found at the edge of defects;8 weeks later,a small amount of periodontal tissue reached its original height and gingival epithelium proliferated obviously.Conclusion Collagen membrane combined with rhBMP-2 which has not only conductive effect but also effect of osteoinduction,can prevent the long-epithelium growing,maintain the growth gap,and promote periodontal tissue to regenerate.
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Objective To investigate the effects of stress associated with nicotine on experimental periodontal breakdown of ligature-induced periodontitis in rats.Methods Forty male Wistar rats with the neck of left maxillary second molar ligated by nylon were used.The animals were randomly divided into four groups:Ni group,0.7 mg of nicotine?kg-1?d-1 (intraperitoneal); S group,stress (immobilization-12 h?d-1); Ni+S group,stress (immobilization-12 h?d-1) associated with an intraperitoneal injection of 0.7 mg of nicotine?kg-1?d-1; C group:saline solution. Two rats of each group were sacrificed at the day of 2,4,6,8,10 separately and the furcation areas of the second maxillary molars were examined histologically and histometrically.Results Intergroup analysis revealed a higher bone loss rate in the animals of Ni+S group compared with the other three groups,the absorption rate of alveolar bone at the day of 6,8,10 in experimental groups were higher than that in C group (P
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Objective To investigate the effect of different concenrtrations of basic fibroblast growth factor (bFGF) on proliferation of human dental pulp cell in vitro,and to find out the most effective concentration of bFGF. Methods Dental pulp cells were isolated from dental pulp tissue explants.The pulp cells were divided into 5 groups randomly,bFGF was added into each group until the ultimately concentrations were 0.1,1.0,10.0 and 100.0 ?g?L-1respectively while the group without bFGF as control group. The effects of bFGF on dental pulp cells were assayed by absorbency A and relative growth rate(RGR) with MTT colorimetric method. Results bFGF at concentrations of 1.0-100.0 ?g?L-1 promoted the cell proliferation (P0.05). Conclusion bFGF has the capability of promoting the proliferation of human dental pulp cells,and the smallest effective concentration is 1.0 ?g?L-1,the most strong cell proliferation takes place at bFGF concentration of 10.0 ?g?L-1.
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Objective To establish a suitable animal model of experimental periodontitis and study the relationship between stress and inflammatory periodontal diseases.Methods Forty Wistar rats with nylon thread placed around the neck of maxillary left second molar tooth were divided randomly into two groups:stress group(the rats were treated with restraint strees for 12 h every day for 10 d)and control group.Four rats of either group were sacrificed at the day of 2,4,6,8,10,separately;the level of blood glucose and the contents of adrenocorticotropic hormone(ACTH),orticosterone and adrenaline were measured as the stress markers,as well as the relative weight of the thymus and spleen.The furcation area of the second maxillary molar was examined histologically and histometrically.Results In stress group,all the markers for stress were significant higher than those in control group,and the thymus and spleen were atrophied(P
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Objective:To observe the effects of nicotine on the proliferation and fibronectin(Fn) synthesis of human periodontal ligament cells(PDLCs) cultured in vitro,and to investigate the mechanism of the effects of smoking on the periodontitis.Methods:PDLCs were cultured in the presence of nicotine at various concentrations,the proliferation of cells was measured by MTT chromatometry,and the synthesis of Fn was measured by ELISA and reverse transcription-polymerase chain reaction(RT-PCR).Results:①MTT chromatometry:Nicotine inhibited the proliferation of PDLCs obviously(P