脂多糖诱导外泌体对牙髓干细胞牙源性分化的影响及机制

Effect of exosomes secreted by dental pulp stem cells on the differentiation induced by LPS

目的探讨脂多糖(LPS)诱导分泌的外泌体对人牙髓干细胞(HDPSC)牙源性分化的影响及机制。方法提取、分离及培养HDPSC,采用流式细胞术及Western blot鉴定细胞,观察成牙/成骨、成脂诱导HDPSC多向分化,筛选最适浓度LPS诱导HDPSC分泌外泌体、并提取外泌体,将HDPSC分为阴性对照组(PBS)、正常外泌体HDPSC组(N-HDPSC exo)及LPS诱导HDPSCs分泌的外泌体组(L-HDPSC exo),采用细胞增殖检测(MTT)法和流式细胞术检测各组细胞增殖和凋亡情况,碱性磷酸酶(ALP)染色检测各组HDPSC成骨分化表达,采用实时荧光定量PCR检测各组HDPSCs中ALP、重组人牙本质基质蛋白1(DMP-1)、牙本质涎蛋白(DSP)及核心结合蛋白因子2(RUNX2)成骨标志基因的表达,采用Western blot检测各组HDPSCs中转化生长因子-β(TGFβ1)、转化生长因子β受体(TGFβR1)、磷酸化SMAD家族成员2/3(p-SMAD2/3)、SMAD蛋白家族成员2/3(SMAD2/3)及SMAD蛋白家族成员4(SMAD4)蛋白的表达。结果成功分离培养并鉴定的牙髓干细胞,成脂诱导可见红色发亮脂滴,成骨诱导可见钙化结节;LPS诱导HDPSC分泌的外泌体符合其结构特征并且可被HDPSC所摄取;L-HDPSC exo组较N-HDPSC exo组和PBS组细胞增殖能力增加、凋亡水平下降、ALP染色程度增加(P<0.05),ALP、DMP-1、DSP及RUNX2表达增加(P<0.05),TGFβ1、TGFβR1、p-SMAD2/3、SMAD2/3及SMAD4表达增加(P<0.05)。结论LPS诱导HDPSC分泌的外泌体可促进HDPSC增殖及细胞活力,并可促进其向成牙/成骨向分化,机制可能与调控TGFβ1/SMADs信号通路、参与诱导正常牙髓干细胞牙源性分化有关。

Objective To investigate the effect of exosomes secreted by human dental pulp stem cells(HDPSCs)on the differentiation induced by LPS.Methods Extraction and identification of HDPSCs by flow cytometry and Western blot were implemented to observe teeth formation/osteogenesis and adipogenesis induce multi-directional differentiation of HDPSC.Different lipopolysaccharide(LPS)concentrations was used to screen and extract HDPSCs secrete exosomes for the optimal LPS induction concentration,which were divided into the negative control group(PBS group)and normal exosome HDPSC group(N-HDPSC exo group)and the exosome group secreted by HDPSCs induced by LPS(L-HDPSC exo group).Then the cell proliferation and apoptosis of each group were detected by MTT method and flow cytometry.ALP staining was used to examine the osteogenic differentiation expression of HDPSCs and real-time fluorescence quantitative PCR to detect the expression of ALP,DMP-1,DSP,and RUNX2 osteogenic marker genes in each group.Western blot was used to detect the TGFβ1,TGFβR1,p-SMAD2/3,SMAD2/3,SMAD4 protein expressions.Results The cultured and identified HDPSCs were successfully isolated,and red shiny lipid droplets were visible in adipogenic induction,and calcified nodules were visible in osteogenic induction.Exosomes induced by LPS were in conformity with their structural characteristics and could be taken up by HDPSCs.The L-HDPSC exo group significantly increased cell proliferative capacity,reduced apoptosis levels and increased ALP staining,compared with N-HDPSC exo and PBS groups.ALP,DMP-1,DSP,and RUNX2 expressions increased significantly,TGFβ1,TGFβR1,p-SMAD2/3,SMAD2/3,and SMAD4 expressions increased significantly,with statistical significance(P<0.05).Conclusion LPS-induced exosomes secreted by HDPSCs can promote cell proliferation,viability and their differentiation into odontogenesis/osteogenesis,and their mechanism of action may be involved in the induction of odontogenicity of normal HDPSCs by regulating the TGFβ1/SMADs signaling pathway.