Abstract: To study whether apoptotic extracellular vesicles of umbilical cord mesenchymal stem cells（UCMSC-ApoEVs）can promote tooth extraction healing in mice with type 2 diabetes mellitus（T2DM）and explore its mechanism. Methods    Apoptosis of UCMSCs was induced by Staurosporine and then ApoEVs were isolated. The UCMSC-ApoEVs （PBS suspension）were applied to T2DM mice after tooth extraction （T2DM+ApoEVs group，28 db/db mice）. At the same time，normal group（28 db/m mice）and T2DM group （28 db/db mice）were set up，and an equal amount of PBS was injected into the tooth extraction wound. The healing result was evaluated by stereomicroscope，Micro CT and HE staining. Immunofluorescence staining and real-time fluorescence quantitative PCR were used to identify the gene expression of nucleotide binding oligomerization domain-like receptor protein 3（NLRP3），Casepase-1，Gasdermin D，interleukin（IL）-1β and IL-18. Results    The rates of mucosal healing and new bone formation in T2DM group were significantly lower than those in normal group. The final healing bone volume fraction（BV/TV），trabecular thickness（Tb.th）and trabecular number（Tb.N）decreased significantly，and the infiltration of inflammatory cells in submucosa was obvious（P < 0.05）. The mucosal healing and new bone formation of tooth extraction in T2DM+ApoEVs group were significantly accelerated  than that in T2DM group，BV/TV，Tb.th and Tb.N were significantly up-regulated，and the infiltration of inflammatory cells was alleviated（P < 0.05）. The results of immunofluorescence showed that there was obvious colocalization of pyroptosis protein and F4/80 positive cells around the new bone of tooth extraction wound in 3 groups of mice，and the relative fluorescence intensity of pyroptosis protein in T2DM group was higher than that in normal group，which decreased in UCMSC-ApoEVs group （P < 0.05）. The results of PCR showed that the expression of pyroptosis-related protein，IL-1β and IL-18 mRNA in the tooth extraction wound in T2DM group were significantly higher than that in normal group，and decreased significantly after UCMSC-ApoEVs treatment（P < 0.05）. Conclusion   UCMSC-ApoEVs can reduce inflammation by inhibiting the pyroptosis of macrophages，and finally improve the mucosa and bone healing of tooth extraction in T2DM mice.

Key words: type 2 diabetes mellitus, tooth extraction, umbilical cord mesenchymal stem cells, apoptotic extracellular vesicles, pyroptosis

摘要： 目的    研究脐带间充质干细胞凋亡囊泡（apoptotic extracellular vesicles of umbilical cord mesenchymal stem cells，UCMSC-ApoEVs）能否促进2型糖尿病（type 2 diabetes mellitus，T2DM）小鼠拔牙创愈合并探讨其机制。方法    通过星胞菌素诱导UCMSCs凋亡，将提取的UCMSC-ApoEVs（PBS悬液）用于T2DM小鼠拔牙创（T2DM+ApoEVs组，db/db小鼠28只）；另设置正常组（db/m小鼠28只）和T2DM组（db/db小鼠28只），拔牙创注入等量PBS。应用体视显微镜、Micro CT、HE染色等方法评估拔牙创愈合情况，通过免疫荧光染色、实时荧光定量PCR检测拔牙创愈合过程中炎症小体核苷酸结合寡聚化结构域样受体蛋白3（nucleotide binding oligomerization domain-like receptor protein 3，NLRP3）、半胱氨酸蛋白酶（Caspase）-1、Gasdermin家族蛋白D（GSDMD）、白细胞介素（interleukin，IL）-1β、IL-18等巨噬细胞焦亡相关指标。结果    T2DM组小鼠拔牙创黏膜愈合和新骨形成速率均显著低于正常组小鼠，最终愈合的骨体积分数（BV/TV）、骨小梁厚度（Tb.th）、骨小梁数目（Tb.N）显著下降，黏膜下层炎细胞浸润明显（P < 0.05）；T2DM+ApoEVs组小鼠拔牙创黏膜愈合和新骨形成均较T2DM组显著加速，BV/TV、Tb.th、Tb.N显著上调，炎细胞浸润减轻，差异均具有统计学意义（P < 0.05）。免疫荧光结果显示，3组小鼠拔牙创新生骨周围均存在明显的NLRP3、激活态Cleaved Caspase-1、GSDMD与巨噬细胞标志物F4/80共定位；T2DM组焦亡蛋白相对荧光强度高于正常组，而T2DM+ApoEVs组较T2DM组明显下调（P < 0.05）。实时荧光定量PCR结果显示，T2DM组拔牙创内的NLRP3、GSDMD以及IL-1β、IL-18的mRNA表达水平较正常组明显增高，而T2DM+ApoEVs组较T2DM组明显下调（P < 0.05）。结论    UCMSC-ApoEVs可通过抑制巨噬细胞焦亡减轻炎症，最终促进T2DM小鼠拔牙创黏膜及骨愈合。 

关键词: 2型糖尿病, 牙拔除术, 脐带间充质干细胞, 凋亡囊泡, 焦亡