脂多糖预处理的牙囊细胞外泌体对牙周炎牙周膜细胞成骨分化的影响

Exosomes derived from lipopolysaccharide⁃preconditioned dental folic cells regulate osteogenic differentiation of periodontal ligament cell in periodontitis

目的探究脂多糖(lipopolysaccharide,LPS)预处理牙囊细胞(dental folic cells,DFCs)外泌体(exo⁃somes,Exos)在不同浓度条件下对牙周炎患者的牙周膜细胞(periodontal ligament cells of periodontitis,p⁃PDLCs)成骨分化能力的影响,为牙周病的防治提供基础。方法组织块酶消化法培养DFCs及p⁃PDLCs,提取经250 ng/mL LPS刺激DFCs 24 h后的Exos,Exos的表征通过透射电镜扫描、粒径分析以及蛋白印记法进行鉴定;通过逆转录⁃聚合酶链反应(reverse transcription⁃polymerase chain reaction,RT⁃PCR)、茜素红染色等方法检测10μg/mL以及100μg/mL浓度的Exos对p⁃PDLCs成骨向分化能力的影响。结果LPS预处理的DFCs外泌体(L⁃Exos)为直径30~100 nm之间的囊泡样结构,高表达CD63和相互作用蛋白X(ALG⁃2 interacting protein⁃X,Alix)。100μg/mL L⁃Exos上调p⁃PDLCs骨膜蛋白、Ⅰ型胶原、Ⅲ型胶原基因的表达(P<0.05);而10μg/mL L⁃Exos仅上调p⁃PDLCs的转化生长因子⁃β1(transforming growth factor⁃β1,TGF⁃β1)基因的表达(P<0.01)。在成骨诱导7 d后,L⁃Exos组的矿化结节明显多于对照组(P<0.01),并且100μg/mL L⁃Exos组的矿化效果更佳(P<0.01)。结论100μg/mL L⁃Exos较10μg/mL L⁃Exos更有利于增强p⁃PDLCs成骨向分化能力。

Objective The purpose of this study was to investigate the effect of different concentrations of exosomes(Exos)secreted from dental folic cells(DFCs)preconditioned with lipopolysaccharide(LPS)on the osteogenic differentia⁃tion ability of periodontal cells in periodontitis(p⁃PDLCs)in patients to provide a basis for the prevention and treatment of periodontal disease.Method Tissue block and enzyme digestion methods were used to culture DFCs and p⁃PDLCs.Exosomes were isolated from 250 ng/mL LPS⁃preconditioned DFCs 24 h later.The characteristics of exosomes were de⁃tected by transmission electron microscopy,particle size analysis and Western blotting.The effects of 10μg/mL and 100μg/mL exosomes on the osteogenic differentiation of p⁃PDLCs were detected by RT⁃PCR and Alizarin red staining.Results LPS⁃pretreated DFC⁃derived exosomes(L⁃Exos)are vesicle⁃like structures with a size between 30⁃100 nm that positively express CD63 and Alix.Compared with the control group,exosomes significantly upregulated Periostin,ColⅠ,and ColⅢexpression at 100μg/mL(P<0.05),while TGF⁃β1 was significantly upregulated at 10μg/mL(P<0.01).At 7 days after osteogenic induction,mineralized nodules were significantly more abundant in the exosome group than in the control group(P<0.01),and the results were better at a concentration of 100μg/mL(P<0.01).Conclusion 100μg/mL L⁃Exos are better than 10μg/mL L⁃Exos in enhancing the osteogenic differentiation ability of p⁃PDLCs.