| Ginkgo biloba extract(GBE) has widely application in clinical,pharmacy and dentistry. Considerable evidence suggests GBE can elimination of free radicals, adjustment of the microcirculation, improving hemodynamics, and reduces inflammation. However little is known about mechanism that how GBE prevent and treat periodontal diseases.In this study, we want to demonstrate that GBE function through protection of human periodontal ligament cells(PDLCs),which possess differentiation potential and play an important role in the periodontium reconstruction.Objective:To investigate the protective effects of GBE on human PDLCs in vitro, to elucidate the mechanism of GBE on periodontosis,Methods:1. Primarily culture and identify the human PDLCs and observation on biological characteristics of human PDLCs. Through modified tissue culture method, we cultured PDLCs in vitro and identified the cells by immunohistochemical staining.2.①The effects of GBE on the proliferation activity and cell cycle of human PDLCs. The 4-7th passage human PDLCs were digested by 0.25% trypsin. Cells were seeded in 96 well plates at a concentration of 1x104cells/ml.Then removed the culture medium and added the culture medium containing different doses GBE (1,0.1,0.01,0.001mg/ml) after 24h.The culture medium without GBE was used as control. Methyl thiazolil tetracolium(MTT) method was used to observe the proliferation of human PDLCs at 12,24,48,72h.②Different GBE concentrations on human PDLCs, then observe and analyze the phase changes of DNA synthesis and cell cycle in human PDLCs by flow cytometry(FCM).3.①The cells from passage 5 were divided into five test groups at random. Blank control group only added DMEM with 10%FBS, Lipopolysaccharide(LPS) group added 100μg/ml LPS in DMEM with 10%FBS,drug group added GBE of different density(0.1,0.01mg/ml) and 100μg/ml LPS in DMEM with 10%FBS,dexamethasone group added 100μg/ml LPS and 2μg/ml dexamethasone in DMEM with 10%FBS.Cultured for 12,24,48,72h.Observed the growth of human PDLCs of each group by MTT staining and spectrophotometric assay.②The cells from passage 5 were divided into five test groups at random. Blank control group only added DMEM with 10%FBS,LPS group added 100μg/ml LPS in DMEM with 10%FBS,drug group added GBE of different density(0.1,0.01mg/ml) and 100μg/ml LPS in DMEM with 10%FBS,dexamethasone group added 100μg/ml LPS and 2μg/ml dexamethasone in DMEM with 10%FBS.After 12,24,48,72h,we gathered the culture medium separately to detect the secretion level of TNF-a,IL-6,IL-1βby Enzyme linked immunosorbent assay(ELISA) test kit.Results:1. The results show that primary passage human PDLCs dissociated from the tissue after 7 days. The 4-7th passage cells were identified with immunhistochemiacl stainting and the results were:cytokerain(-) and vimentin(+),the majority of human PDLCs had a spindle-shaped,elongated appearance characteristic of fibroblast-like cells in light microscope, the minority were a star-shaped. By HE staining their plasma was pink and blue or purple nucleus,which was round or oval at the center of cells.2. The MTT assay revealed that GBE at lmg/ml could suppress proliferation activities of human PDLCs significantly in comparison with control(P<0.05),GBE at 0.1mg/ml could improve proliferation activities of human PDLCs significantly in comparison with control at 12,24,48h (P<0.05), GBE at 0.01mg/ml could improve proliferation activities of human PDLCs significantly in comparison with control at 24,48,72h (P< 0.05).GBE at 0.001mg/ml couldn't improve proliferation activities of human PDLCs significantly in comparison with control(P>0.05). FCM revealed that the proliferation index(Prl) of GBE at 0.lmg/ml in comparison with control at 12,24,48h (P<0.05), PrI of GBE at 0.01mg/ml in comparison with control at 24,48,72h (P<0.05).3. The MTT assay revealed that LPS at 100μg/ml could suppress the proliferation activities of human PDLCs significantly in comparison with control(P< 0.05),improved greatly after adding 0.1,0.01mg/ml GBE in comparison with LPS group(P<0.05).ELISA assay revealed that LPS at 100μg/ml could improve greatly IL-6,L-1β,NF-αproduction of human PDLCs significantly in comparison with control(P<0.05), after adding 0.1,0.01mg/ml GBE could significantly inhibit the activity of IL-6,L-1β,TNF-a production of human PDLCs stimulated with LPS in comparison with LPS group(P<0.05).Conclusion:1. The results proved that the human PDLCs derived from mesoblast and not epithelium. Successfully cultured human PDLCs which can be used in the further experimentation.2. Different concentration on GBE can affect proliferation on human PDLCs.1mg/ml GBE could suppress human PDLCs proliferation.0.1,0.01mg/ml GBE could improve proliferation of human PDLCs, FCM results demonstrate that GBE reduce number of cells in G1 phase, and increase number of cells in S phase, therefore significantly increased Pr1(S+G2/M)%(P<0.05). As a result, cells proliferation was promoted.3. GBE has the protective effects on human PDLCs via inhibit LPS, a potent stimulator for human PDLCs to generate TNF-a,IL-6,IL-1β,these cytokines are known as pro-inflammatory factors. Thus we propose GBE suppress inflammation through reduce level of TNF-α,IL-6,IL-1β. |
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