摘要
目的探究脐带间充质干细胞外泌体(hUC-MSCs-sEV)调控Notch通路对炎症微环境下人牙周膜干细胞成骨分化的影响。方法体外分离培养人牙周膜干细胞(hPDLSCs),将其随机分为对照组、模型组、hUC-MSCs-sEV低剂量组、hUCMSCs-sEV高剂量组、空载质粒组、h UC-MSCs-sEV高剂量+Notch1敲低组,除对照组外的其余各组以10μg/ml脂多糖处理细胞构建炎症微环境,诱导细胞炎症模型。碱性磷酸酶(ALP)及茜素红染色检测成骨分化;酶联免疫吸附法(ELISA)检测上清液前列腺素E2(PGE2)、白细胞介素(IL)-6、IL-10水平;实时荧光定量PCR与免疫印迹法检测Notch1及成骨相关因子Runt相关转录因子2(Runx2)、骨钙素(OCN)、ALP表达。结果与对照组相比,模型组细胞PGE2、IL-6水平升高,ALP阳性细胞相对比例、矿化结节相对含量、IL-10水平、Notch1及Runx2、OCN、ALP表达降低(P<0.05);与模型组相比,hUC-MSCs-sEV低、高剂量组PGE2、IL-6水平依次降低,ALP阳性细胞相对比例、矿化结节相对含量、IL-10水平、Notch1及Runx2、OCN、ALP表达依次升高(P<0.05);敲低Notch1可逆转高剂量hUC-MSCs-sEV对上述指标的影响(P<0.05)。结论hUC-MSCs-sEV可减轻hPDLSCs炎症,促进其成骨分化,其作用机制可能与激活Notch信号通路有关。
To investigate the influence of umbilical cord mesenchymal stem cell exosomes(hUC-MSCs-sEV)on the osteogenic differentiation of human periodontal ligament stem cells(hPOLSCs)in inflammatory microenvironment by regulating Notch pathway.hPDLSCs were isolated and cultured in vitro,and then randomly grouped into control group,model group,low-dose hUC-MSCs-sEV group,high-dose hUC-MSCs-sEV group,empty plasmid group,high-dose hUC-MSCs-sEV+Notch1 knockdown group.Except for the control group,the other groups were treated with 10μg/ml lipopolysaccharide to build an inflammatory microenvironment and induce a cellular inflammation model.The osteogenic differentiation was detected by alkaline phosphatase(ALP)and alizarin red staining;the levels of prostaglandin E2(PGE2),interleukin(IL)-6 and IL-10 in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA);the expressions of Notch1 and osteogenesis-related factors Runtrelated transcription factor 2(Runx2),osteocalcin(OCN)and ALP were detected by real-time quantitative PCR and Western blotting.Compared with the control group,the levels of PGE2 and IL-6 in the model group increased,while the relative proportion of ALP-positive cells,the relative content of mineralized nodules,the level of IL-10,the expressions of Notch1,Runx2,OCN and ALP decreased(P<0.05);compared with the model group,the levels of PGE2 and IL-6 in the low and high dose hUC-MSCs-sEV groups decreased successively,while the relative proportion of ALP-positive cells,the relative content of mineralized nodules,the level of IL-10,the expressions of Notch1,Runx2,OCN and ALP increased successively(P<0.05);knocking down Notch1 can reverse the effect of high-dose hUC-MSCs-sEV on the above indexes(P<0.05).In conclusion,hUC-MSCs-sEV can reduce the inflammation of hPDLSCs and promote their osteogenic differentiation,and the mechanism may be related to the activation of Notch signaling pathway.
作者
李长宏
钟霞
黄琼
LI Changhong;ZHONG Xia;HUANG Qiong(Department of Periodontology,Xining Stomatological Hospital,Xining 810000,China;Department of Orthodontics,Xining Stomatological Hospital,Xining 810000,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2023年第8期662-671,共10页
Immunological Journal
关键词
脐带间充质干细胞外泌体
Notch
炎症微环境
人牙周膜干细胞
成骨分化
Umbilical cord mesenchymal stem cell exosome
Notch
Inflammatory microenvironment
Human periodontal ligament stem cells
Osteogenic differentiation
