Résumé
Objective: To explore suitable concentration of recombinant human transforming growth factor β1 (rhTGF-β1) usage and study the effect of rhTGF-β1 on differentiation of dental pulp stem cells (DPSCs).Methods: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro.Different concentrations (1 , 6 , 10 μg/L) of rhTGF-β1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected.For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-β1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum.The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice.Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate.The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid (BCA) protein assay kit].To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader.The mean difference was considered significant at 0.05 and 95% confidence interval.Results: The DPSCs had ty-pical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days.6 μg/L rhTGF-β1 significantly promoted the DPSCs proliferation on the 3rd and 5th days.After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 μg/L rhTGF-β1 increased ALP activities compared with the control;D values in the 6 μg/L rhTGF-β1 group was 0.31±0.03, while the control group was 0.02±0.01(P<0.05).The total protein content in the 6 μg/L rhTGF-β1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05).To eliminate the cells proliferation influence, relative ALP activities, which was defined as the total ALP divided by the total protein content, the 6μg/L rhTGF-β1 group was 6 times higher than the control group.Alizarin red S staining showed increased mineral nodule formation in the rhTGF-β1 group.The elution of staining under microplate reader also showed more optical density in the 6 μg/L rhTGF-β1-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05).Conclusion: 6 μg/L rhTGF-β1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.
Résumé
AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3βand protein phosphatase (PP) 2A in the rats induced by amyloid βprotein 25-35 (Aβ25-35) in combination with AlCl3 and re-combinant human transforming growth factor ( RHTGF)-β1( composited Aβ) .METHODS:The male SD rats were used to establish the simulated Alzheimer disease ( AD) model by intracerebroventricular injection of composited Aβ.The Morris water maze was applied for screening the successful model rats with learning and memory deficits .The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids ( GLF) at 140 mg/kg for 37 d.The silver nitrate staining was used to determine the cortical NFT .The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3βand PP2A in hippocampus and cortex were determined by Western blot .The mRNA expression of GSK3βand PP2A in the hippocampus and cortex was detected by RT-PCR.RESULTS:Compared with sham group , the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased .The protein levels of phosphorylated tau protein at Ser 199 and Ser214 sites, GSK3βin the hippocampus and cerebral cortex in the model rats dramatically elevated , and PP2A was marked decreased as compared with the sham group rats.Meanwhile, the mRNA expression of GSK-3βsignificantly increased but PP2A was de-creased.However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d.CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3βand PP2A, thus reducing the phosphorylation of tau protein .