大肠杆菌脂多糖诱导人乳牙牙周膜干细胞炎症模型的建立

Establishment of inflammation model of human deciduous periodontal ligament stem cells induced by Escherichia coli lipopolysaccharide

目的体外培养人乳牙牙周膜干细胞,建立符合实验设计需要的人乳牙根尖周炎细胞模型。方法采用酶解组织块法培养人乳牙牙周膜干细胞,连续梯度稀释法进行纯化,流式法鉴定细胞表面标记物,检测其多向诱导分化能力。在培养基中加入不同终浓度(0.1,0.075,0.05,0.025,0.01μg/ml)的大肠杆菌脂多糖,通过检测细胞活性及炎症因子表达,选择合适的大肠杆菌脂多糖浓度。结果酶解组织块法可获得成集落状生长的人乳牙牙周膜干细胞,流式鉴定高表达CD146、STRO-1、CD105、CD90、CD29,低表达CD45、CD34,细胞成骨、成脂诱导后分别对茜素红、油红O染色阳性。0.05μg/ml的大肠杆菌脂多糖不影响细胞增殖活性(P>0.05),能显著提高炎症因子IL-1β、TNF-α、IL-6的mRNA和蛋白表达水平(P<0.05)。结论0.05μg/ml的大肠杆菌脂多糖可作为诱导人乳牙牙周膜干细胞炎症模型的适宜浓度。

Objective To establish a model of inflammation of periodontal ligament stem cells in deciduous teeth.Methods Human periodontal ligament stem cells in deciduous teeth were cultured by enzyme tissue block method,and isolated and purified by serial dilution culture method.Then the cell surface markers were detected by flow cytometry,and the osteogenic and adipogenic differentiation induction abilities were analyzed.The periodontal ligament stem cells of human deciduous teeth were treated with different concentrations of E.coli LPS(0.1,0.075,0.05,0.025,0.01μg/ml),and the cell proliferation and the expression of inflammatory factors were detected to screen the suitable concentration of E.coli LPS for the model establishement.Results Periodontal ligament stem cells in deciduous teeth were obtained using enzyme tissue block method.Cells highly expressed mesenchymal stem cell surface markers including CD146,STRO-1,CD105,CD90,CD29,and lowly expressed vascular endothelial cell surface markers including CD45 and CD34.After multiple induction culture,the cells were positive for alizarin red staining and oil red staining.The 0.05μg/ml E.coli LPS did not significantly affect the cell proliferation(P>0.05),but significantly increased the mRNA and protein expression levels of IL-1β,IL-6 and TNF-α(P<0.05).Conclusion The 0.05μg/ml E.coli LPS is a suitable concentration to induce the inflammation model of human deciduous periodontal ligament stem cells.