Abstract: Objective    To explore the therapeutic effect of exosomes derived from stem cells from apical papilla（SCAP-Exo）on Sjögren syndrome（SS）mice. Methods    SCAP were primarily cultured and identified by enzymatic digestion. SCAP-Exo were isolated by ultracentrifugation and identified by transmission electron microscopy，nanoparticle tracking analysis and western blotting. Six 10-week-old female ICR mice were selected as the control group；twelve 10-week-old female NOD mice were randomly divided into NOD group and SCAP-Exo group，with six in each group. After 2 and 4 weeks of rearing，the mice in the SCAP-Exo group were injected with SCAP-Exo through the tail vein，and the mice in the other groups were injected with PBS through the tail vein. After 6 weeks of rearing，the saliva flow rate of each group of mice was measured；then peripheral blood was collected. The ratio of T helper 17（Th17）/CD4+ T cells in peripheral blood was detected by flow cytometry，and the expression of interleukin-17A（IL-17A）in serum was detected by ELISA. Finally the mice were sacrificed，and the level of lymphocyte infiltration in the submandibular gland was observed by HE staining. Results    Transmission electron microscopy imaging showed that SCAP-Exo was cup-shaped and nanoparticle tracking analysis indicated that the diameters of SCAP-Exo ranged approximately from 30 to 150 nm. The specific marker protein of exosome CD9 and Alix was positively expressed，but Calnexin was not expressed. The overall comparison of saliva flow rate，peripheral blood Th17/CD4+ T cell ratio and IL-17A expression level of the three groups of mice showed statistically significant differences （F values were 59.169，293.217，and 189.583，respectively，all P < 0.05）. Compared with the control group，the saliva flow rate of NOD group was significantly reduced，while the peripheral blood Th17/CD4+ T cells ratio and IL-17A expression levels were significantly increased；compared with the NOD group，the saliva flow rate of SCAP-Exo group mice was increased significantly，while the ratio of Th17/CD4+ T cells in peripheral blood and IL-17A expression level were decreased significantly；the differences were statistically significant（all P < 0.05）. HE staining showed that，compared with the NOD group，the infiltrating level of submandibular gland lymphocytes of SCAP-Exo group were decreased，and only mild scattered lymphocyte infiltration or few lymphocyte infiltration foci could be observed. Conclusion    SCAP-Exo has a superior therapeutic effect on SS mice，which may be related to the regulation of Th17 cells.

Key words: stem cells from apical papilla, exosome, Sj?gren syndrome, T helper 17

摘要： 目的    探索根尖牙乳头干细胞来源的外泌体（exosomes derived from stem cells from apical papilla，SCAP-Exo）对舍格伦综合征（Sjögren syndrome，SS）小鼠的治疗效果。方法    酶消化法提取根尖牙乳头干细胞，超速离心法提取SCAP-Exo，并应用透射电镜、纳米颗粒跟踪技术及Western Blot进行鉴定。选择10周龄雌性健康ICR小鼠6只作为对照组；选择10周龄雌性NOD小鼠12只随机分为NOD组和SCAP-Exo组，每组6只。饲养2、4周后，SCAP-Exo组小鼠尾静脉注射SCAP-Exo，其他组小鼠尾静脉注射PBS。于饲养6周后检测各组小鼠唾液流率。随后收集小鼠外周血，流式细胞术检测外周血中辅助性T细胞17（T helper 17，Th17）/CD4+ T细胞比例，ELISA检测血清中白细胞介素-17A（interleukin-17A，IL-17A）表达水平。最终处死各组小鼠，通过HE染色观察下颌下腺中淋巴细胞浸润水平。结果    透射电镜下可见SCAP-Exo呈杯状的囊泡结构；纳米颗粒跟踪技术分析SCAP-Exo直径为30 ~ 150 nm；外泌体特异性标记蛋白CD9、Alix呈阳性表达，不表达细胞内质网特异性蛋白Calnexin。3组小鼠唾液流率、外周血Th17/CD4+ T细胞比例及IL-17A表达水平总的比较，差异均有统计学意义（F值分别为59.169、293.217、189.583，均P < 0.05）。其中，与对照组相比，NOD组小鼠唾液流率明显降低、外周血Th17/CD4+ T细胞比例及IL-17A表达水平明显升高；而相较于NOD组，SCAP-Exo组小鼠唾液流率明显增加、外周血Th17/CD4+ T细胞比例及IL-17A表达水平明显降低；差异均有统计学意义（均P < 0.05）。相较于NOD组，SCAP-Exo组小鼠下颌下腺淋巴细胞浸润水平显著降低，仅存在轻度散在的淋巴细胞浸润或出现极个别的淋巴细胞浸润灶。结论    SCAP-Exo能有效治疗SS小鼠，可能与其调节Th17细胞转化有关。

关键词: 根尖牙乳头干细胞, 外泌体, 舍格伦综合征, 辅助性T细胞17