| Objective:Paradentosis has a high incidence among the diabetic patients, and leads to severe damage rapidly, paradentosis is one of the major complications of diabetes. It has been confirmed that advanced glycation end products (AGEs) have increased levels of accumulation in the blood of diabetic patients and may delay the repair of periodontal tissue's damage, that means AGEs may play a important role in the occurrence of paradentosis and diabetes. At present, it is known that lipopolysaccharide(LPS) is one of the most important inflammatory factors of periodontal tissue. The effect of LPS in occurrence and development of paradentosis is becoming more and more important. Periodontal ligament stem cell(PDLSC) originates from the periodontal ligament of adult stem cells, having the ability of self replication, can produce different types of mature cells that have specific phenotype and function, also can maintain the stability of function of periodontal ligament, have a important effect on maintain the renew of normal periodontal tissue and the rerepair of periodontal tissue's damage. Therefore, this research set PDLSCs Cultured in vitro as model. We obversed the effects of AGEs on the proliferation and multipotentiality of HPDLSCs mediated by LPS or LPS stimulated monocyte under the supernate, in order to study the influence of diabetes on formation and development of paradentosis.Methods:Culture of primary HPDLSCs using tissue explants collagenase digestion method. Limiting dilution method was used for screening and purification of HPDLSCs. By measuring cloning efficiency and osteogenic and adipogenic differentiation capacity and using immunofluorescence staining and flow cytometry methods detected cell phenotype in order to identify HPDLSCs. Proliferation of HPDLSCs in (Normal medium)C group, (10μg/ml and 100μg/ml LPS) LPS group, (50μg/ml,100μg/ml and 200μg/ml AGEs)AGEs group, (10μg/ml LPS&100μg/ml AGEs)LA group were analyzed by MTT assay. The expression of osteoblast gene(ALP, BSP, RUNX-2) and adipogenic gene(LPL and PPAR-y) in C group, LA group, (10μg/ml LPS) L group, (Monocyte supernatant stimulated by lOng/ml LPS) F group, (Monocyte supernatant stimulated by 10ng/ml LPS&100μg/ml AGEs)FA1 group, (Monocyte supernatant stimulated by lOng/ml LPS&200μg/ml AGEs) FA2 group were detected by Real Time-PCR.Results:1 Identification of HPDLSCs:1.1 Cloning efficiency of HPDLSCs is (32±1.21) %.1.2 HPDLSCs can form mineralized nodules in osteogenic induction and can form lipid droplets under the adipogenic induction.1.3 Relatively specific markers of STRO-1 mesenchymal stem cells were detected by immunofluorescence cytochemistry methods, staining result shows positive.1.4 Results of identification of cell phenotype shows: FITC-CD34-(1.7%), CD14-(1.9%), CD29+(99.6%), CD105+(98.6%), PE-CD146+(52.6%), PECY5-CD45-(1.5%). It means cells isolated in vitro display the characteristics of stem cells.2 MTT results:2.1 Proliferation of HPDLSCs were inhibited by LPS with different concentrations compared with the C group, the proliferation of HPDLSCs were statistically significant (p<0.05).2.2 Proliferation of HPDLSCs were inhibited by AGEs with different concentrations compared with the C group, the proliferation of HPDLSCs were statistically significant (p<0.05) inhibited by AGEs(100μg/ml) and AGEs(200μg/ml), compared with AGEs(50μg/ml).2.3 Proliferation of HPDLSCs were inhibited in L group and LA group, compared with C group. The inhibition of the latter is more obvious than the former (p<0.05)3 Real time-PCR results:After differentiated into osteoblasts for 7 days:(1) the expression of osteoblast gene(ALP, BSP, RUNX-2) were decreased in L group and LA group, compared with C group, but the expression of osteoblast gene of the former were significantly decreased than the latter. (2) the expression of osteoblast gene were decreased in F group and increased in FA1 group, compared with C group. (3) the expression of osteoblast gene were decreased in F group and FA2 group, but the expression of osteoblast gene of the latter were significantly decreased than the former. After differentiated into adipocytes for 7 days, the expression of adipocytes gene(LPL and PPAR-y) were decreased in L group and the expression of adipocytes gene were increased LA group, compared with C group. After differentiated into osteoblasts for 14 days, the expression of osteoblast gene were decreased in L group and LA group, compared with C group, but the expression of osteoblast gene of the latter were significantly decreased than the former. After differentiated into adipocytes for 14 days, the expression of adipocytes gene were increased in L group and LA group, compared with C group, but the expression of adipocytes gene of the latter were significantly increased than the former.Conclusion:1 HPDLSCs isolated in vitro displays the characteristics of stem cells, including Cloning ability, self-renewal and multilineage differentiation capacity.2 Different kinds of cytokines or different concentrations of cytokines can reduce the proliferation of HPDLSCs in a concentration-dependent manner. So we speculated that diabetes-related factors AGEs can inhibit the proliferation of cells in the presence of periodontitis, leading to reduction of self-renewal of periodontal tissue.3 After induced to mineralization for 7 days, HPDLSCs whatever induced by LPS or induced by LPS-induced monocyte supernatant can both inhibit the differentiation into osteoblasts, the differentiation into osteoblasts can be improved when adding 100μg/ml diabetes-related factors AGEs, but the differentiation into osteoblasts can be inhibited when adding 200μg/ml diabetes-related factors AGEs. After differentiated into adipocytes for 7 days, HPDLSCs stimulated by LPS can lead to inhibition of differentiation into adipocytes, the inhibition of differentiation into adipocytes can be improved when adding 100μg/ml diabetes-related factors AGEs. We speculate that 100μg/ml diabetes-related factors AGEs can increase periodontitis tissue's ability to repair in the way of promoting periodontal. ligament stem cells into the adipogenic and osteoblasts differentiation, but 200μg/ml diabetes-related factors AGEs can inhibit periodontitis tissue's ability to repair in the way of inhibiting periodontal ligament stem cells into the osteoblasts differentiation.4 In 2 weeks, HPDLSCs stimulated by LPS can lead to inhibition of differentiation into osteoblasts, enhancement of differentiation into adipocytes. HPDLSCs induced by LPS can both inhibit the differentiation into osteoblasts, the differentiation into osteoblasts and adipocytes can be restrained when adding 100μg/ml diabetes-related factors AGEs. We speculate that 100μg/ml diabetes-related factors AGEs can lead to periodontitis tissue damage, leading to increased inflammation. Diabetic patients with periodontitis have increased inflammation may be through influencing the biological characteristics of periodontal ligament stem cell to influencing self-renewal and self-repair capacity of periodontitis inflammatory tissue. |
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