牙髓干细胞来源凋亡小体调节巨噬细胞极化及炎症反应

Dental pulp stem cell⁃derived apoptotic bodies regulate macrophage polarization and inflammatory response

目的探讨牙髓干细胞(dental pulpstemcells,DPSCs)释放的凋亡小体(apoptotic bodies,ABs)对巨噬细胞极化及体内炎症反应的调节作用。方法分离培养、鉴定人来源DPSCs并利用星孢菌素诱导其凋亡,对ABs进行表征鉴定。将体外培养的巨噬细胞分为对照组、LPS组以及LPS+ABs组,分别施加溶剂对照处理、LPS处理、LPS和ABs共处理,观察巨噬细胞对ABs的吞噬情况以及各组M型巨噬细胞标志物CD206表达及细胞因子释放水平的差异。构建小鼠皮肤创伤模型及小鼠葡聚糖硫酸钠结肠炎模型,分为PBS组、DPSCs组及ABs组,分别注射PBS对照、DPSCs及DPSCs来源的ABs,观察各组小鼠体重、损伤局部组织形态、CD206表达、组织再生及细胞因子表达等情况的差异。结果分离培养的DPSCs表面标志物及分化潜能符合间充质干细胞特征。DPSCs在凋亡过程中释放的ABs符合典型凋亡小体特征。ABs可被体外培养的巨噬细胞吞噬,并提高LPS处理组巨噬细胞CD206表达、降低其促炎因子肿瘤坏死因子⁃α(tumor necrosisfactor⁃α,TNF⁃α)和白细胞介素⁃6(interleukin⁃6,IL⁃6)的释放,同时增加炎症调节因子转化生长因子⁃β(transforming growthfactor⁃β,TGF⁃β)的释放(P<0.01)。在皮肤创伤模型中,尾静脉注射ABs显著提高皮肤缺损愈合速度(P<0.05),降低创伤局部炎性因子表达(P<0.01),并提高CD206阳性细胞数量(P<0.01);在结肠炎模型中,ABs有效维持小鼠体重(P<0.05)和结肠长度(P<0.01),并显著增加局部CD206阳性细胞数量(P<0.01)。结论DPSCs释放的ABs可促进M2型巨噬细胞极化并调节炎性反应,有望替代活细胞移植应用于炎症调节及组织再生。

Objective To investigate the effects of apoptotic bodies(ABs)derived from dental pulp stem cells(DP⁃SCs)on macrophage polarization and inflammation response in vivo.Methods Human DPSCs were extracted,cultured and identified.Staurosporine was used to apoptosis induction and differential methods were performed for ABs identifi⁃cation.The in vitro cultured macrophages were divided into 3 groups:solvent control,lipopolysaccharide(LPS),and the LPS+ABs.The macrophages were stimulated with LPS to induce inflammation followed by ABs treatment.In the untreat⁃ed group,macrophages were added with an equal amount of solvent.The specific uptake of ABs by macrophages,the ex⁃pression level of CD206 and the levels of inflammatory cytokines were analyzed.The mouse models of cutaneous wounds and dextran sulfate sodium(DSS)⁃induced colitis were established,and the mice were randomly divided into 3 groups:the PBS⁃treated group,the DPSCs⁃treated group,and the ABs⁃treated group.The mice were injected with the same volume of PBS,DPSCs and ABs,respectively.The body weight,histological pathology,the expression levels of CD206 and cytokines,and the extent of tissue regeneration were measured.Results DPSCs and ABs derived from DP⁃SCs were successfully isolated and characterized.ABs could be taken up by macrophage.While lipopolysaccharide(LPS)induced production of tumor necrosis factor⁃α(TNF⁃α)and interleukin⁃6(IL⁃6),ABs significantly reduced the levels of these pro⁃inflammatory cytokines and increased the expression of transforming growth factor⁃β(TGF⁃β)and CD206(P<0.01).In the cutaneous inflammatory wound model,the wound closure rate in mice intravenously injected with ABs was significantly accelerated(P<0.05).The administration of ABs markedly reduced the pro⁃inflammatory factors levels and increased the CD206+cell number.In the colitis model,treatment with ABs markedly reduced the loss in bodyweight(P<0.05),recovered the colon length(P<0.01),and significantly increased the CD206+cell num⁃ber.Conclusion DPSCs⁃derived ABs could enhance macrophage M2 polarization and attenuate inflammation.There⁃fore,ABs could be used as a promising cell replacement for inflammatory regulation and tissue regeneration.