Effect of aspirin on cell biological activities in murine bone marrow stromal cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine the effect of aspirin on cell proliferation, alkaline phosphatase (ALP) activity, cell cycle and apoptosis in murine bone marrow stromal cells, so as to explore an appropriate dose range to improve bone regeneration in periodontal treatment.</p><p><b>METHODS</b>ST2 cells were stimulated with aspirin (concentrations of 1, 10, 100 and 1 000 μmol/L) for 1, 2, 3, 5 and 7 d. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. After ST2 cells were treated for 1, 3 and 7 d, ALP activity was measured by ALP kit, cell cycle and apoptosis were measured by flow cytometry (FCM) after treated for 48 h.</p><p><b>RESULTS</b>MTT assays showed that various doses of aspirin have different effects on the cell growth. Briefly, lower concentrations (1, 10 μmol/L) of aspirin promoted the cell growth, the A value of 0, 1 and 10 μmol/L aspirin 7-day-treated cells were 0.313±0.012, 0.413±0.010 and 0.387±0.017 respectively (P <0.01 vs control), and so did the ALP level ([4.3±0.9], [6.0±0.3] and [7.7±0.4] μmol·min(-1)·g(-1), P <0.05 vs control), while higher concentrations, especially 1000 μmol/L of aspirin might inhibit the cell growth with time going, A value and ALP level were 0.267±0.016, (4.3±1.3) μmol·min(-1)·g(-1) respectively (P <0.05 vs control). Cell cycle analysis revealed no changes in comparison to control cells after treatment with 1 or 10 μmol/L aspirin, but it was observed that cell mitosis from S phase to G2/M phase proceeded at higher concentrations of 100 μmol/L aspirin, and the cell cycle in phase G0/G1 arrested at 1000 μmol/L. Parallel apoptosis/necrosis studies showed that the percentage of cells in apoptosis decreased dramatically at all doses of aspirin, the apoptosis rates of ST2 cells responded to 0, 1, 10, 100 and 1000 μmol/L aspirin were (11.50±0.90)%, (5.30±0.10)%, (5.50±0.10)%, (4.90±0.90)% and (7.95±0.25)% respectively (P<0.05 vs control).</p><p><b>CONCLUSIONS</b>This study demonstrated that lower dosage of aspirin can promote ST2 cells growth, osteogenic activity and inhibit its apoptosis. Aspirin maybe used for the bone reconstruction with a proper concentration.</p>

Culturing and characterization of human gingival mesenchymal stem cells and their chemotactic responses to stromal cell-derived factor-1 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4 in human gingival mesenchymal stem cells (GMSCs) and the migration potential of GMSCs stimulated with SDF-1.</p><p><b>METHODS</b>Human GMSCs were isolated by single-cell cloning method. Their cell surface markers were characterized by flow cytometry, and the rate of colony formation was evaluated. Differentiation assay was used to detect the differentiation potential of GMSCs. The expression of chemokine SDF-l receptor CXCR4 in GMSCs was detected by immunocytochemical staining. The chemotactic effect of SDF-1 on GMSCs was detected using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.</p><p><b>RESULTS</b>Human GMSCs possessed high self-renewal potential and formed single-cell colonies cultured in vitro. GMSCs expressed mesenchymal stem cells-associated markers CD44, CD73, CD90, CD105, and CD166, and the expression of hemopoietic stem cell surface markers CD14, CD34, and CD45 was negative. GMSCs differentiated into osteoblasts and adipocytes under defined culture conditions. The colony forming unit-fibroblastic for GMSCs was 21.4%/±2.8%. Immunocytochemical staining demonstrated that GMSCs expressed chemokine SDF-1 receptor CXCR4. The number of GMSCs migrating at concentrations of 100 ng.mL-1 and 200 ng.mL-1 of SDF-l in the Transwell cell culture chamber was significantly higher than that of the negative control (189.3±4.4, 164.6±4.9 cells/field vs. 47.8±2.5 cells/field, P<0.01). Treatment with the CXCR4 neutralizing antibody, an antagonist for CXCR4, significantly reduced the migratory effect compared with the negative controls (29.0±2.4 cells/field vs. 47.8±2.5 cells/field, P<0.01).</p><p><b>CONCLUSION</b>Human GMSCs express chemokine SDF-l receptor CXCR4. SDF-1 may participate in regulating chemotaxis of human GMSCs. Results suggest that the migration induced by SDF-1 is mediated by CXCR4.</p>

Expression of high mobility group box 1 in gingival tissues of chronic periodontitis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of high mobility group box 1 (HMGB1) in gingival tissues of chronic periodontitis.</p><p><b>METHODS</b>Human peripheral blood mononuclear cells(PBMC) were stimulated with 1 microg x mL(-1) lipopolysaccharide (LPS) for 24 h or 48 h. Expression and release of HMGB1 were checked by immunofluorescence and enzyme-linked immunosorbent assay (ELISA), respectively. PBMC were stimulated with 100 ng x mL(-1) HMGB1 or 50 ng x mL(-1) tumor necrosis factor-alpha (TNF-alpha), the expressions of TNF-alpha and HMGB1 in the supernatant were studied by ELISA. Gingival tissues and gingival crevicular fluids (GCF) were collected from patients and healthy people. Expression of HMGB1 in gingival tissues and GCF was studied using immunofluorescence and ELISA, respectively.</p><p><b>RESULTS</b>HMGB1 was translocated from nucleus to cytosol in PBMC after LPS stimulation for 24 h. The content of HMGB1 in the supernatant from stimulated cells was significantly higher than that from unstimulated cells after 48 h (P < 0.01). HMGB1 was released by PBMC in response to TNF-alpha stimulation, it also stimulated PBMC to release TNF-alpha (P < 0.01). Translocation of HMGB1 from nucleus to cytosol was also found in infiltrated cells in gingival tissues from patients, and HMGB1 in GCF from patients was significantly higher than that from healthy people P < 0.01).</p><p><b>CONCLUSION</b>The results suggest that HMGB1 may play an important role in the pathological progress of chronic periodontitis.</p>

Mechanism of carbon monoxide affecting the expression of cellular adhesion molecule under stimulation of inflammatory cytokines to human gingival fibroblasts / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism by which carbon monoxide inhibits the expression of adhesion molecules on human gingival fibroblasts (HGF) stimulated with inflammatory cytokines.</p><p><b>METHODS</b>HGF were cultured in vitro, and stimulated with 50 ng x mL tumor necrosis factor-alpha (TNF-alpha) and 10 ng x mL(-1) interleukin-1beta (IL-1beta) concurrently in the presence or absence of carbon monoxide releasing molecule-3 (CORM-3) at 500 micromol x L-1. Expression of phosphorylated extracellular regulated protein kinase (ERK), phosphorylated c-Jun N-terminal kinase (NK) and phosphorylated p38 in mitogen-activated protein kinase(MAPK) pathway was studied by Western blot at 10 min and 20 min, respectively. Nuclear expression of nuclear factor-kappaB (NF-kappaB) was checked by Western blot after 4 h stimulation. In some experiments, cells were prestimulated by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) for 8 h before cytokine stimulation and the expression of intercellular adhesion molecule-1 (ICAM-1) was checked by Western blot after 24 h.</p><p><b>RESULTS</b>CORM-3 significantly inhibited the phosphorylation of MAPK p38 after 10 min stimulation with cytokines, but had no signifi-cant effect on the phosphorylation of ERK and JNK. CORM-3 significantly inhibited the nuclear expression of NF-KB-p65 on HGF after 4 h stimulation by inflammatory cytokines. The inhibitory effect of CORM-3 on the expression of ICAM-1 was not influenced by guanylate cyclase inhibitor ODQ.</p><p><b>CONCLUSION</b>The inhibitory effect of carbon monoxide on the expression of adhesion molecules might be exerted by its inhibitory effect on the NF-kappaB activity and MAPK p38 phosphorylation.</p>

Minocycline hydrochloride liposome controlled-release gel improves rat experimental periodontitis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the therapeutic effects of 2% minocycline hydrochloride liposome controlled-release gel on the periodontitis in an established rat periodontitis model.</p><p><b>METHODS</b>Biocompatibility was tested by oral perfusion sample solution for long-term observation. Minocycline hydrochloride liposome controlled-release gel was utilized to treat the established rat periodontitis model. The rats were selected randomly and divided into three groups: group A (PERIO-treated group), group B (minocycline hydrochloride liposome controlled-release gel treated group), and group C (negative control group). The gingival index (GI) and probing depth (PD) were detected, and the number of mononuclear and broken bone cells were examined after 7, 14, 28, and 56 d.</p><p><b>RESULTS</b>The minocycline hydrochloride liposome controlled-release gel exhibited excellent biocompatibility based on weight measure and tissue section evaluation. The rats with periodontitis demonstrated that GI, PD, and the number of mononuclear and broken bone cells of group B decreased in 14, 28, and 56 d. Pathological observation showed that new bones and fibers were formed in group B.</p><p><b>CONCLUSION</b>Minocycline hydrochloride liposome controlled-release gel improves rat periodontitis, thereby providing valuable evidence for clinical application.</p>

Influence of carbon monoxide on the expression of adhesion molecules stimulated with tumor necrosis factor-alpha and interleukin-1beta on human gingival fibroblasts / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the influence of carbon monoxide on the expression of adhesion molecules stimulated by inflammatory cytokines on human gingival fibroblasts.</p><p><b>METHODS</b>Human gingival fibroblasts were stimulated with 50 ng x mL(-1) tumor necrosis factor (TNF)-alpha and 10 ng x mL(-1) interleukin (IL)-1beta concurrently in the presence or absence of 500 micromol x L(-1) carbon monoxide releasing molecule (CORM). Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 at protein and mRNA level was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Activity of transcription factor NF-kappaB was evaluated by reporter gene assay.</p><p><b>RESULTS</b>Expression of ICAM-1 and VCAM-1 on human gingival fibroblasts increased dramatically after concurrent stimulation of TNF-alpha and IL-1beta, while CORM inhibited the upregulation of ICAM-1 and VCAM-1. CORM decreased the activity of NF-KB stimulated by TNF-alpha and IL-1beta.</p><p><b>CONCLUSION</b>Carbon monoxide could be a promising way in treating of periodontitis.</p>

Culturing and characterization of human periodontal ligament stem cells and investigating their chemotactic responses to bone morphogenetic protein-2 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the chemotactic response of human periodontal ligament stem cells (PDLSCs) to bone morphogenetic protein-2 (BMP-2).</p><p><b>METHODS</b>Human PDLSCs were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate PDLSCs by limited dilution method. The expression of Vimentin and stem cell marker STRO-1 on PDLSCs were demonstrated with immunocytochemical staining. Differentiation assay was used to detect the differentiation potential of PDLSCs. Cloning formation experiment and 5-bromo-2-deoxyuridine (BrdU) incorporation assay were used to determine the stem cell characteristics of PDLSCs. The chemotactic effect of BMP-2 on PDLSCs was detected by using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.</p><p><b>RESULTS</b>Human PDLSCs displayed positive staining for Vimentin and expressed the stem cell marker STRO-1. These cells differentiated into osteoblasts and adipocytes under defined culture conditions, possessed high self-renewal potential and formed single-cell colonies in vitro. The number of cells migrating at concentrations of 100, 200 ng mL(-1) of BMP-2 in Transwell cell culture chamber was significantly higher than that of negative control (P<0.01).</p><p><b>CONCLUSION</b>BMP-2 may participate in regulating chemotaxis of human PDLSCs.</p>

Expression of FHL2 during mineralization of human periodontal ligament cells in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression pattern of FHL2, which is an intracellular signaling transcription molecule during mineralization in cultured human periodontal ligament cells (hPDLCs) in vitro.</p><p><b>METHODS</b>hPDLCs were cultured in vitro. Test group was cultured with mineral induction media while control group without induction media. 0, 14, 28 days after culture, alizarin red staining was used to measure the mineral nodules formation. Immunocytochemistry was used to examine the expression of FHL2 protein 0 day and 14 days after mineral induction. Meanwhile, mRNA expression level of FHL2 was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) on the 0, 14, 28 days after induction.</p><p><b>RESULTS</b>14 and 28 days after cultivation, mineral nodules formed and were stained positively with alizarin red staining in test group while no mineral nodule formed in control group. Immunocytochemical results indicated that hPDLCs in test group expressed FHL2 positively. According to RT-PCR results, 14 and 28 days after mineral induction, the expression levels of FHL2 both increased significantly when compared with 0 day (P<0.01), and the expression level at 14 days was 1.4 folds of 0 day.</p><p><b>CONCLUSION</b>FHL2 protein is found to be involved in the in vitro mineralization of hPDLCs. FHL2 protein may play a role in the differentiation and mineralization of hPDLCs.</p>

Effects of exogenous IL-10 on IL-6 and ICAM-1 expression in inflammatory gingival tissue / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at investigating effects of exogenous interleukine-10 (IL-10) on IL-6 and intercellular adhesion molecular (ICAM-1) expression in inflamed gingival tissue.</p><p><b>METHODS</b>The expression of IL-6 and ICAM-1 was examined using immunohistochemical techniques. Inflammatory gingival tissue treated with IL-10 was taken as the experimental group and the same patient's inflammatory gingival tissue without treatment of IL-10 was included into the control group.</p><p><b>RESULTS</b>IL-6 expression was found mainly in monocytes, macrophages, lymphocytes, endotheliocytes and fibroblasts. The expression of ICAM-1 was found mainly in epithelial cells, monocot-macrophages, lymphocytes, endotheliocytes and fibroblasts. The immunohistochemical optical density (IOD) of the expression of IL-6 and ICAM-1 was detected by using Image-Proplus software, and the results showed that the expression in the experimental group differed significantly from that in the control group.</p><p><b>CONCLUSION</b>The exogenous IL-10 may down-regulate IL-6 and ICAM-1 expression in inflammatory gingival tissue.</p>

Effects of IGF-1 on the biological characteristics of mouse dental follicle cells / 实用口腔医学杂志

Objective:To study the effects of IGF-1 on the proliferation,total protein amount and cytodifferentiation of mouse dental follicle cells.Methods:The dental follicle cells of passage 3 were incubated with different concentrations((0.005),0.01,0.05,0.1 and 0.5 mg/L)of IGF-1 respectively,cell proliferation,alkaline phosphatase(ALP),total protein amount were measured by MTT assay and spectrophotometer respectively.Effects of 0.05 and 0.1 mg/L IGF-1 on mineralization potential were studied by Von Kossa staining.Results:IGF-1(ml/L) at 0.005~0.1 increased the proliferation and total protein amount(P

Construction of a vector expressing SBR gene / 实用口腔医学杂志

Objective: To obtain a prokaryotic expression vector containing saliva binding region (SBR) gene of Streptococcus mutans. Methods: By directional cloning method, SBR gene fragment was cloned into the expression vector pcMVT7, the recombinant plasmid pcMVT7-SBR was transformed to E.coli JM109 (DE3). The gene expression was induced with IPTG. Restriction endonuclease and DNA sequencing techniques were used to identify the recombinant plasmid DNA, and finally target protein was purified by affinity chromatography. Results:The DNA sequence of SBR in the reconstructed vector pcMVT7-SBR was in corresponding with the initial design. The C-terminal 6?His tagged SBR fusion protein was expressed in JM109(DE3) and was purified by affinity chromatography. The expression rate of target protein was 29.73%. Conclusion:The recombinant expression plasmid pcMVT7-SBR was constructed.