目的:探讨根尖牙乳头干细胞(stem cells from apical papilla, SCAP)摄取牙髓干细胞(dental pulp stem cells, DPSCs)外泌体的作用,为揭示其内吞外泌体的途径提供依据。方法:(1)采用超速离心法结合超滤法提取DPSCs外泌体,采用透射电镜观察法、纳米粒子示踪分析法以及Western Blot对其进行鉴定。(2)采用PKH-26膜标记技术标记DPSCs外泌体,37 ℃条件下将其与SCAP共培养作为阳性对照组,4 ℃条件下将其与SCAP共培养作为低温处理组,同时设置阴性对照组。采用免疫荧光染色法观察不同培养温度条件下SCAP内红色荧光标记情况。(3)通过胞吞抑制方法观察SCAP摄取外泌体的胞吞途径,分别采用10 μmol/L氯丙嗪(chlorpromazine,CPZ,抑制网格蛋白介导的胞吞途径)作为CPZ组、200 μmol /L 金雀异黄素(genistein,抑制小窝蛋白介导的胞吞途径)作为Genistein组、50 μmol /L LY294002抑制巨胞饮(macropinocytosis)作用作为LY294002组处理SCAP,将PKH-26标记的DPSCs外泌体与SCAP共培养,同时设置溶剂对照组(添加与抑制剂组等量的DMSO),采用免疫荧光染色技术观察SCAP内红色荧光标记情况和流式细胞技术分析有红色荧光标记的SCAP百分比。结果:(1)DPSCs外泌体形态呈茶托样,具有双层膜结构,粒径峰值为144 nm,能够表达肿瘤易感基因(tumor susceptibility gene、TSG)101蛋白、CD63蛋白,二者皆为外泌体标志蛋白,符合外泌体特征。(2)免疫荧光结果显示,37 ℃共培养6 h后可见SCAP内有大量红色荧光(PKH-26)标记,而4 ℃共培养6 h后,SCAP内未见明显红色荧光(PKH-26)标记。(3)免疫荧光结果显示胞吞抑制后,SCAP内部红色荧光(PKH-26)标记减少,流式结果显示阳性对照组红色荧光标记的SCAP占35.0%,阴性对照组红色荧光标记的SCAP占0.5%,溶剂对照组红色荧光标记的SCAP占29.7%,CPZ组、Genistein组、LY294002组分别下降至13.7%、 16.6%、 20.9%。结论:SCAP能够摄取DPSCs外泌体,低温可影响该摄取过程;SCAP摄取外泌体主要依赖网格蛋白介导的胞吞途径、小窝蛋白介导的胞吞途径以及巨胞饮途径。

Objective: To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. Methods: (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 ℃ as positive control group, and co-cultured with SCAP at 4 ℃ as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluore-scence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluore-scence. Results: (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 ℃ for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 ℃ for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%,and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. Conclusion: SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.