摘要
目的:探究乳牙牙髓干细胞(SHED)来源凋亡囊泡(ApoVs)通过水解细胞外促炎介质腺苷三磷酸(ATP)缓解结肠炎的分子机制。方法:取牙髓组织培养SHED细胞并完成鉴定;星形孢菌素诱导SHED细胞凋亡后,利用梯度离心法提取ApoVs,通过粒径分析、电镜扫描和Western blot方法进行鉴定;通过活体成像检测ApoVs靶向小鼠炎症组织能力;利用葡聚糖硫酸钠构建结肠炎小鼠模型,尾静脉注射磷酸盐缓冲液(PBS)、ApoVs或外核苷酸焦磷酸酶1(ENPP1)抑制剂预处理ApoVs治疗,通过小鼠体质量变化、结肠长度及HE染色检测治疗效果;收集小鼠血清检测ATP水平。通过Western blot检测ApoVs中ENPP1水平,构建体外酶促反应系统验证SHED来源ApoVs介导水解ATP的分子机制。结果:成功培养SHED细胞及SHED来源ApoVs并完成鉴定;活体成像显示ApoVs能够靶向小鼠结肠组织;构建结肠炎小鼠模型并观察各组治疗效果,结果显示ApoVs治疗组小鼠结肠长度得到明显恢复(P<0.05),HE组织评分显著降低(P<0.05),血清ATP浓度明显降低(P<0.05);而预处理ApoVs抑制其ENPP1蛋白功能后,治疗小鼠体质量、肠道长度及HE组织评分改善效果降低(P<0.05),小鼠血清ATP浓度降低不明显(P<0.05)。Western blot结果显示SHED ApoVs中大量富集ENPP1蛋白(P<0.05);体外酶促系统验证结果显示ApoVs能够水解SHED细胞ATP,且水解能力随着ApoVs浓度的升高而增强(P<0.05);使用抑制剂ENPP1-IN预处理ApoVs后,ApoVs水解SHED细胞ATP的能力下降(P<0.05)。结论:SHED来源的ApoVs通过表达ENPP1水解促炎介质ATP,抑制炎症反应从而缓解结肠炎。
Objective:To explore the potential mechanism by which apoptotic vesicles(ApoVs)derived from stem cells from human exfoliated deciduous teeth(SHED)alleviate colitis by hydrolyzing extracellular pro-inflammatory mediators.Methods:The dental pulp tissue was collected to culture SHED cells followed by characterization;after inducing SHED cell apoptosis with staurosporine,ApoVs were extracted using gradient centrifugation and identified through particle size analysis,scanning electron microscopy,and Western blot;in vivo imaging was performed to assess the targeting ability of ApoVs to inflammatory tissues;a dextran sulfate sodium(DSS)-induced colitis mouse model was established,and the therapeutic effects were evaluated by tail vein injection of phosphate-buffered saline,ApoVs or ENPP1 inhibitor-pretreated ApoVs,with assessments including histological scoring,colon length,and body weight changes,while serum was collected to measure ATP levels;Western blot was employed to determine ENPP1 levels in ApoVs,and an in vitro enzymatic reaction system was constructed to verify the role of mediating ATP hydrolysis by ApoVs.Results:SHED cells were successfully cultured and their markers were identified;in vivo imaging demonstrated that ApoVs could target colon tissue;in vivo experiments revealed that,compared to the model group,mice in the ApoVs treatment group exhibited significant recovery in colon length(P<0.05),a notable reduction in histological scores(P<0.05),and a marked decrease in serum ATP concentration(P<0.05);when ApoVs were pretreated to inhibit their ENPP1 protein function,the therapeutic effects(body weight,colon length,and histological scores)were significantly attenuated(P<0.05),and the reduction in serum ATP concentration was not significant(P<0.05).Western blot results showed that ENPP1 protein expression levels in ApoVs were significantly elevated(P<0.05);the results of the in vitro enzymatic system verification showed that ApoVs could hydrolyze the ATP of SHED cells,and the hydrolytic ability increased with the increase of ApoVs concentration(P<0.05);after pretreatment with the inhibitor ENPP1-IN,the hydrolytic ability of ApoVs to hydrolyze the ATP of SHED cells decreased(P<0.05).Conclusions:SHED-derived ApoVs alleviate colitis by expressing ENPP1 to hydrolyze ATP,the pro-inflammatory mediator,thereby suppressing the inflammatory response.
作者
范思远
周洋
李子涵
杨晓姗
轩昆
刘世宇
伍美玲
FAN Siyuan;ZHOU Yang;LI Zihan;YANG Xiaoshan;XUAN Kun;LIU Shiyu;WU Meiling(State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi Clinical Research Center for Oral Diseases,Department of Preventive Dentistry,School of Stomatology,The Fourth Military Medical University,Xi'an 710032,China;State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi Key Laboratory of Stomatology,Department of Oral Biology,School of Stomatology,The Fourth Military Medical University,Xi'an 710032,China)
出处
《口腔生物医学》
2025年第5期248-254,267,共8页
Oral Biomedicine
基金
国家自然科学基金青年项目(82201013,82301099)。
关键词
乳牙牙髓干细胞
凋亡囊泡
外核苷酸焦磷酸酶1
ATP
结肠炎
stem cells from human exfoliated deciduous teeth
apoptotic vesicles
ectonucleotide pyrophosphatase phosphodiester-ase 1
ATP
colitis
