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Objective@# To explore the effects of red LED light mediated by the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (KEAP1-NRF2/HO-1) pathway on osteogenic differentiation and oxidative stress damage of human periodontal ligament stem cells (hPDLSCs) induced by high glucose, which provides a basis for the application of red light-emitting diode (LED) light in cell antioxidative damage.@*Methods@#hPDLSCs were identified by flow cytometric analysis, alkaline phosphatase (ALP) staining and Alizarin red-S staining; hPDLSCs were pretreated in a high glucose environment for 48 hours and irradiated with 1, 3, or 5 J/cm2 red LED light. A CCK-8 assay was performed to choose the radiant exposure that had the strongest effect on promoting the cell proliferation rate for subsequent experiments. hPDLSCs were divided into a control group, a high glucose group and a high glucose+light exposure group. ALP staining, ALP activity, Alizarin red-S staining and quantitative calcified nodules were used to detect the osteogenic differentiation of hPDLSCs; qRT-PCR and Western blot were used to detect the gene and protein expression levels of ALP, runt-related transcription factor 2 (RUNX2) and osterix (OSX); the relative mRNA expression levels of antioxidant enzyme-related genes superoxide dismutase 2 (SOD2) and catalase (CAT) in hPDLSCs were detected by qRT-PCR; reactive oxygen species (ROS) levels were detected by fluorescence microscopy and flow cytometry; the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in cell supernatants were detected by ELISA; the NRF2-specific inhibitor ML385 was used to inhibit the NRF2 pathway; ALP staining and ALP activity were used to detect the markers of early osteogenic differentiation; qRT-PCR was used to detect the gene expression of ALP, RUNX2 and OSX; and the protein expression levels of KEAP1, NRF2 and HO-1 were detected by Western blot.@*Results @# Identified, and irradiant exposure of 5 J/cm2 was chosen for subsequent experiments. Red LED light irradiation (5 J/cm2) improved the osteogenic differentiation of hPDLSCs induced by high glucose (P<0.05), increased the mRNA and protein levels of ALP, RUNX2 and OSX (P<0.05), upregulated the mRNA expression levels of SOD2 and CAT (P<0.05), reduced the levels of ROS (P<0.05), and reduced TNF-α and IL-1β levels in the cell supernatants (P<0.05). When ML385 was added to inhibit the NRF2 pathway, the ALP activity of cells was decreased (P<0.05); the gene expression levels of ALP, RUNX2 and OSX were downregulated (P<0.05); the protein level of KEAP1 was upregulated (P<0.05); and the protein levels of NRF2 and HO-1 were downregulated (P<0.05)@*Conclusion@#Red LED light may promote the proliferation and osteoblastic differentiation of hPDLSCs induced by high glucose through the KEAP1-NRF2/HO-1 pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.
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OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.
Subject(s)
Humans , Anti-Inflammatory Agents/pharmacology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chemokine CXCL12 , Lipopolysaccharides/pharmacology , Osteogenesis , Periodontal Ligament/metabolism , Receptors, Chemokine/metabolism , Stem Cells , Interleukin-8/metabolismABSTRACT
BACKGROUND: The conditioned medium rich in bioactive substances can maintain the stability of proliferation and biological characteristics of stem cells. Whether the conditioned medium of human periodontal stem cells derived from healthy tissues can affect the proliferation and osteogenesis of human periodontal stem cells derived from inflammatory tissue is significant for periodontal tissue regeneration and reconstruction. OBJECTIVE: To investigate the effect of human periodontal stem cells-conditioned medium derived from healthy tissue on proliferation and osteogenic differentiation of human periodontal stem cells derived from inflammatory tissue. METHODS: HPDLSCs from normal periodontal ligaments of healthy adults were isolated, purified and cultured in vitro. Human periodontal stem cells-conditioned medium was obtained by collecting the supernatants from the serum free medium which was used for the third generation cells grown up to 80% of the bottom of the bottle after 24 hours cultivation. Human periodontal stem cells derived from inflammatory tissue were obtained from pericementum of periodontitis patients, and cultured by using limiting dilution assay. Human periodontal stem cells derived from inflammatory tissue were separately cultured under conditioned medium treatment group (conditioned medium containing 50% human periodontal ligament stem cells + 50% conventional medium) and control group (conventional medium). Protein expression levels of vimentin, Pan Cytokeratin, and stromal cell antigen STRO-1 were identified by immunofluorescence staining. The proliferative activity of cells was analyzed by MTT assay and flow cytometry. After osteogenesis in vitro, alkaline phosphatase activity and the expression levels of three osteogenesis related genes (Runx2, OPN, and OCN) were detected using alkaline phosphatase kit and RT-PCR, respectively in both groups. RESULTS AND CONCLUSION: (1) Both groups of cells were in accordance with the morphological characteristics of adult stem cells, showing long fusiform or polygonal shapes. There was no significant difference in cell morphology between the two groups under inverted phase contrast microscope. (2) The immunofluorescence staining showed that cells in both groups were positive for the specific antibodies of vimentin and STRO-1, but negative for the specific antibody of Pan Cytokeratin. (3) The results of MTT assay showed that after 3, 5 and 7 days, the proliferative activity of conditioned medium treatment group was higher than that of control group (P < 0.01). (4) Cell cycle analysis showed that compared with the control group, the number of cells in G2/M phase and S phase in conditioned medium treatment group increased significantly (P < 0.05). (5) At 5 and 7 days of osteogenic induction in vitro, alkaline phosphatase activity of conditioned medium treatment group was higher than that of control group (P < 0.05). (6) After 21 days of osteogenic induction, the expression levels of osteogenic related genes Runx2, OPN, and OCN in conditioned medium treatment group were significantly higher than those in control group (P < 0.01, P < 0.05). (7) In conclusion, human periodontal stem cells-conditioned medium derived from healthy tissues can enhance the proliferation and osteogenic differentiation of human periodontal stem cells derived from inflammatory tissue.
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OBJECTIVES@#This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs).@*METHODS@#The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment.@*RESULTS@#The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (@*CONCLUSIONS@#Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.
Subject(s)
Humans , Cell Differentiation , Cell Proliferation , MicroRNAs/genetics , Osteogenesis , Periodontal Ligament/cytology , Potassium , Potassium Channels, Voltage-Gated , RNA, Long Noncoding/genetics , Stem Cells/cytologyABSTRACT
OBJECTIVE@#To explore the mechanism of Piezo1 protein in mediating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) via the Notch signaling pathway.@*METHODS@#In this study, young permanent teeth extracted from impacted teeth of 8-14-year- old children from January 1, 2016 to January 1, 2018 in the Department of Orthodontic, Beijing Children's Hospital were selected as cell sources. hPDLSCs were extracted by enzymatic digestion. Immunohistochemical staining was used to detect the expression of keratin and vimentin, and flow cytometry was used to identify the markers (CD146 and STRO-1) of hPDLSCs. The construction and screening of Piezo1 siRNA gene interference vector and Piezo1 gene overexpression plasmid were completed. Flexcell 4000T mechanical distraction stress instrument was used to construct hPDLSC cell model in vitro. According to the preliminary results, the experiment was divided into five groups: siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of Piezo1, Notch1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and bone sialoprotein (BSP). Western blot was used to detect the expression of ALP and Runx2. Fluo-3 AM probe was used to detect intracellular calcium content.@*RESULTS@#Vimentin staining of hPDLSCs was positive, and keratin staining was negative. Flow cytometry was used to detect the expression of STRO-1 and CD146, markers of hPDLSC. Empty viral vectors, siRNA-Piezo1 interference sequence, and Piezo1 overexpression vector sequence could be transfected into hPDLSC by lentivirus, and the transfection efficiency was high (approximately 90%). The reverse transcription-polymerase chain reaction (RT-PCR) results showed that there were significant differences in Piezo1 gene levels among the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=9.573, P<0.05). The level of Piezo1 in the overexpression group was significantly higher than that in the siRNA interference group (q=3.893, P<0.05). The level of Piezo1 in the stretch stress group was significantly higher than that in the blank control group (q=2.006, P<0.05). The expression of Notch1 and osteogenic genes ALP, Runx2, OCN, and BSP had the same trend. Western blot results showed that there were significant differences in the expression of ALP in the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=11.207, P<0.001). The expression level of ALP in the overexpression group was significantly higher than that in the siRNA interference group (q=2.991, P<0.05). The expression of ALP in the stretch stress group was significantly higher than that in the blank control group (q=3.007, P<0.05). The expression of Runx2 protein showed the same trend. The intracellular calcium fluorescence intensity of the overexpression group was significantly higher than that of the siRNA interference group, and the intracellular calcium fluorescence intensity of the stretch stress group was significantly higher than that of the siRNA interference group.@*CONCLUSIONS@#Mechanical stretch stress can promote the expression of Piezo1 protein. Ca2+ is the second messenger, activates the Notch1 signaling pathway and the expression of ALP, Runx2, OCN, and BSP; and promotes the osteogenic differentiation of hPDLSC. The siRNA-Piezo1 interfering plasmid can block this process. On the contrary, the overexpression plasmid of Piezo1 can promote the osteogenic differentiation of PDLSCs.
Subject(s)
Child , Humans , Alkaline Phosphatase , Cell Differentiation , Cells, Cultured , Ion Channels , Osteogenesis , Periodontal Ligament , Signal Transduction , Stem CellsABSTRACT
Objective @#To investigate the effects of casein kinase 2 interacting protein-1 (CKIP-1) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs).@*Methods @#The hPDLSCs were obtained by primary culture with periodontal ligament tissues that were collected from normal humans. Then, a lentiviral vector containing a CKIP-1-specific siRNA sequence was constructed, and the transcriptional level of CKIP-1 in hPDLSCs was downregulated after vector infection. The P4 cells were divided into four groups: the control group, negative control group (infected with a control vector), CKIP-siRNA group (infected by a CKIP-1 siRNA lentivirus) and CKIP-1 group (infected by a CKIP-1 overexpression virus). All of the cells were cultured under osteogenic induction for 21 days. Then, alizarin red staining and quantitative determination were performed to detect the osteogenic differentiation ability of the hPDLSCs. In addition, qPCR was used to detect the transcriptional level of osteogenesis-related regulatory factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL), and the osteogenesis-related regulatory factors of the bone morphogenetic protein (BMP) signaling pathway.@*Results@#There were no differences in the indexes between the negative control group and the control group (P > 0.05). Compared with the negative control group, the CKIP-siRNA group demonstrated more mineralized nodules (P < 0.05), significantly increased calcium salt deposition (P < 0.05), and increased mRNA levels of osteogenesis-related regulatory factors, such as Runx2 , ALP, OCN, and RANKL, and the osteogenesis-related regulatory factors of BMP signaling pathway (P < 0.05). @*Conclusion@#Downregulation of CKIP-1 could promote the osteogenic differentiation of hPDLSCs, which is related to the transcription level of osteogenic-related regulatory factors.
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Objective: To investigate the effect of calcitonin (CT) on promoting collagen synthesis and osteogenesis of human periodontal ligament stem cells (hPDLSCs). Methods: Fifty adult participants were divided into chronic periodontitis (CP) group (n=25) and control group (n=25). In the CP group, the anterior maxilla with probing depth ≥5 mm and the sites with imaging evidence of bone loss were selected. The gingival crevicular fluid (GCF) samples were collected from 6 maxillary sites in each patient. In the control group, multiple sites without inflammation (10 to 12 per subject) were sampled to ensure that a sufficient amount of GCF was collected. The expression of CT, transforming growth factor β1 (TGF-β1) and bone morphogenetic protein (BMP) 2/4/7 in GCF was detected by enzyme linked immunosorbent assay (ELISA), and the correlation between CT expression and clinical parameters such as periodontal pocket probing depth (PD), clinical attachment loss (CAL) and gingival index (GI), and the above-mentioned indicators was investigated with Spearman correlation analysis. hPDLSCs were infected with the adenoviruses carrying CT gene (Ad.CT) and the expression of mRNA and protein of TGF-β1, BMP2/4/7, alkaline phosphatase (ALP), osteocalcin (OCN) and collagen type I/III (Col I/III) were detected by quantitative real-time PCR and Western blotting. Results: The expression level of CT in GCF of the CP group was significantly higher than that of the control group ([32.62±1.46] ng/mL vs [17.70 ± 0.76] ng/mL, P<0.01). The expression of CT was positively correlated with clinical parameters such as PD, CAL and GI (P<0.01, P<0.05). The expression levels of BMP2/4/7 and TGF-β1 in GCF of the CP group were significantly higher than those of the control group (BMP2: [138.67 ± 4.04] ng/mL vs [103.96 ± 2.78] ng/mL, BMP4: [155.53 ± 3.55] ng/mL vs [133.15 ± 2.92] ng/mL; BMP7: [106.59 ± 2.85] ng/mL vs [90.22±1.56] ng/mL; TGF-β1: [105.92 ± 3.40] ng/mL vs [89.85 ± 2.42] ng/mL; all P<0.01). The expression of BMP2/4/7 and TGF-β1 was negatively correlated with CT expression (P<0.01, P<0.05). The overexpression of CT significantly increased the expression of TGF-β1, Col I/III and osteoblast markers BMP2/4, ALP and OCN in GCF (all P<0.01). Compared with the cells co-infected with Ad.CT and Ad.Null, the cells co-infected with Ad.CT and small interfering RNA specifically blocking TGF-β1 (Ad.TGF-β1 siRNA) had significantly lower collagen expression (Col I: 0.16 ± 0.02 vs 0.22 ± 0.03; Col III: 0.11±0.01 vs 0.15 ± 0.02; both P<0.01). Compared with Ad.CT infected cells, the protein expression levels of ALP and OCN were significantly decreased in Ad.CT and noggin co-treated cells (ALP: 0.19 ± 0.02 vs 0.25 ± 0.03; OCN: 0.13 ± 0.01 vs 0.19 ± 0.02; both P<0.01). Conclusion: CT can promote collagen synthesis and osteogenesis in hPDLSCs through TGF-β1 and BMP signaling transduction pathways.
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Objective@#To investigate the effect of overexpression of Notch intracellular domain (NICD) on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSC).@*Methods@#The third generation hPDLSC with stable overexpressing of NICD were assigned as experimental group, normal hPDLSC were as negative control group and hPDLSC transfected with empty vector were as blank control group. The effect of overexpressing NICD on proliferation ability of hPDLSC was detected by using cell counting kit-8 (CCK-8). Alizarin Red staining and real-time quantitative PCR (qPCR) were used to detect the effects of NICD on cementum attachment proteins (CAP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and Notch signal pathway receptor Notch1. The effect of overexpressing NICD on hPDLSC osteogenic protein RUNX2 and flag marker protein (used to label NICD) were detected by using Western blotting.@*Results@#CCK-8 results showed that there were no significant differences in A values amongst the three groups for 1-2 days (P>0.05). The number of cells in the experimental group was significantly increase than that of the two control groups from the third to seventh days (A values were 0.203±0.016, 0.364±0.014, 0.449±0.020, 0.549±0.020 and 0.570±0.020, respectively) (P<0.05). Alizarin red staining showed that compared with the blank control group and negative control group, the mineralized nodules in the experimental group had smaller formation range and lighter color, and the differences were statistically significant (P<0.05). The expressions of CAP gene (0.751±0.058, 0.887±0.025), osteocalcin gene (0.592±0.051, 0.670±0.045) and RUNX2 gene (0.319±0.038, 0.684±0.055) at 14 and 21 days in the experimental group were significantly lower than those in the negative control group respectively (P<0.05). However, the expression levels of Notch1 gene at 14 and 21 days (2.507±0.047, 4.041±0.219) were significantly higher than those of negative and blank control groups (P<0.05). The results of Western blotting showed that the expressions of flag marker protein (0.167±0.007, 0.204±0.010) at 14 and 21 days in the experimental group were significantly higher than those in the negative and blank control groups (P<0.05). However, the expressions of RUNX2 protein (0.075±0.006, 0.074±0.013) at 14 and 21 days were significantly lower than that in the negative control group (0.092±0.003, 0.118±0.008) and blank control group (0.174±0.006, 0.212±0.008) (P<0.05).@*Conclusions@#Overexpression of NICD can promote the proliferation capacity of hPDLSC and inhibit its osteogenic differentiation.
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Objective To observe the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to investigate the epigenetic regulation of EZH2 inhibitor DZNeP on osteogenic differentiation of hPDLSCs. Methods The hPDLSCs were isolated and cultured, and their proliferation under different concentrations of DZNeP (0, 1, 2, 5 and 10 μmol/L) was detected by MTT. The effects of DZNeP on osteogenic differentiation of hPDLSCs were observed by alkaline phosphatase (ALP) staining and alizarin red staining. The effect of DZNeP on the trimethylation of histone H3K27 in hPDLSCs was detected by immunofluorescence staining. Results Compared with the control group, the proliferation of hPDLSCs after 1, 2, 5 and 10 μmol/L DZNeP treatment for 48 h was significantly decreased, respectively (all P<0.05), and it was concentration-dependent. The result of ALP staining and alizarin red staining showed that DZNeP could promote the expression of early osteogenic markers ALP and the formation of advanced calcified nodules of hPDLSCs. The immunofluorescence staining result showed that the trimethylation fluorescence intensity of histone H3K27 was significantly decreased in the DZNeP group compared with the control group. Conclusions As an EZH2 inhibitor, DZNeP can inhibit the proliferation of hPDLSCs and promote the differentiation of hPDLSCs into osteoblasts in vitro, suggesting that DZNeP can be used as a potential small molecule drug for the treatment of periodontitis.
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OBJECTIVE@#This study aims to compare the osteogenic differentiation capability of stem cells derived from human inflammatory periodontal ligament tissues (iPDLSCs) with those of stem cells derived from healthy periodontal ligament tissues (hPDLSCs). Both types of tissues were induced by stromal cell derived factor (SDF-1) in vitro.@*METHODS@#iPDLSCs and hPDLSCs were primarily cultured by tissue digestion method and purified by limited dilution cloning. The cells were passaged and identified by stem cell surface marker expression through flow cytometry. Then, we used thiazolyl blue tetrazolium bromide to detect and compare the proliferation capabilities of the iPDLSCs and hPDLSCs. Express of bone volumes were detected by alizarin red staining after SDF-1 was added to the cells. Using alkaline phosphatase, we evaluated the osteogenic differentiation capability of the cells induced by SDF-1. The expression levels of the osteogenesis-related genes of the cells induced by SDF-1 were determined by reverse transcription-polymerase chain reaction.@*RESULTS@#After purification, both iPDLSCs and hPDLSCs expressed stem cell markers. hPDLCSs had a higher proliferation capability than iPDLSCs. Osteogenesis-related genes had higher expression levels in the cells induced by SDF-1 than in those without induction (P<0.05). SDF-1 at 50 and 200 ng·mL⁻¹ concentration greatly affected the differen-tiation capabilities of iPDLSCs and hPDLSCs respectively.@*CONCLUSIONS@#iPDLSCs and hPDLSCs had osteogenic differentia-tion capability. The level of osteogenic differentiation in normal and inflamed periodontal ligament stem cells increases after SDF-1 induction.
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Humans , Cell Differentiation , Cell Proliferation , Cells, Cultured , Osteogenesis , Periodontal Ligament , Stem Cells , Stromal CellsABSTRACT
Objective To investigate the effect of β-catenin on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) under mechanical tension.Methods PDLSCs were seperately cultured in vitro from the periodontium attached at freshly extracted teeth.β-catenin-targeting agonist or inhibitor was applied to the PDLSCs to upregulate or downregulate the expression of activity of β-catenin protein.The expression of β-catenin protein and the osteogenetic related markers(ALP,BMP2,Runx 2) under the mechanical tension with different period(0,6,12,24 h) were assessed with Western blot in the PDLSCs irritated with agonist or inhibitor.Results Compared with the PDLSCs without mechanical tension,the expression of osteogenesis related marker proteins,ALP and Runx 2,in PDLSCs were enhanced under the circular mechanial tensile stress (P ＜ 0.05).In the PDLSCs irritated with WAY-262611,the β-catenin-targeting agonist,the expression of osteogenetic related markers ALP in the PDLSCs was improved during the first 12 h period of mechnical tensile load,which was significantly higher than that in the DMSO group.Conclusion Wnt/β-catenin signaling pathway plays an important role in the early osteogenesis differentiation of PDLSCs under cyclic tension.The β-catenin promotes osteogenic differentiation of PDLSCs under mechanical tension.
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Objective To explore the effect of Notch1 signal pathway on the osteogenic differentiation in human periodontal ligament stem cells(PDLSCs) under dynamic strain.Methods PDLSCs were separated from freshly extracted teeth then identified and prolifed.Notch1 signal pathway was regulated by chemicals.Dynamic strains were applied to PDLSCs with the tension plus system.Then Notch1 signal pathway key factor Notch intracellular domain(NICD),osteoblastic related indexes alkaline phosphatase(ALP) and bone morphogenetic proteins 2 (BMP2) were detected by western blot expression.The deformation rate of stress parameters was 0 to 12%,and the frequency was 0.1 Hz.The loading time was 0 h,6 h,12 h and 24 h.Results As Notch1 signal pathway was activated,the expression of osteogenic markers ALP and BMP2 both reduced (P<0.05).On the contrary, the expression of osteogenic markers ALP and BMP2 both increased obviously (P<0.05) as Notch1 signal pathway was inhibited.Conclusion The activated Notch1 signal pathway will inhibit osteogenic differentiation of PDLSCs under dynamic tensile.
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Objective To up-regulate the expression of Glil gene in periodontal ligament stem cells ( PDLSCs) and to explore the effect of Glil gene on PDLSCs proliferation and osteogenesis differentiation by establishing Glil gene adenovirus vectors. Methods Subcloned Glil to viral backbone vector Adtrack-CMV and transfered the established vector to 293T cells, which was to acquire the virus particles. Trans-fected aim cells,namely PDLSCs,with these virus. Detected its effect on PDLSCs proliferation with CCK-8 assay, and detected the expression of Glil and the bone-related markers ALP and Runx2 through Western blot. Results An adenovirus vector, which were over expressed Glil gene, was successfully constructed and transfected to PDLSCs. Compared with the empty vector group and normal group, the over expressed one had a much slower proliferation rate in CCK-8 assay (P=0. 003). Western blot showed that ALP and Runx2 can be overexpressing os-teogenic differentiated after PDLSCs successfully transfected with the Glil gene. Conclusion Over expressing Glil gene would lead to a much slower proliferation rate in the PDLSCs and an increase of the bone-related markers. It is concluded that Glil can enhance the osteogenic dif-ferentiation capacity in PDLSCs.