Objectives: The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs.
Methods: Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-β (TGF-β), were detected using real-time quantitative polymerase chain reaction (qRT-PCR).
Results: Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-β and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05).
Conclusions: The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.
目的: 通过将Cal27外泌体（Cal27-exo）与正常人牙龈成纤维细胞（NHGFs）共培养，测定共培养后NHGFs+Cal27-exo的增殖迁移能力及相关蛋白表达情况，探讨Cal27-exo对NHGFs的活化及生物学行为的影响。方法: 采用超速离心法提取Cal27-exo，并用蛋白免疫印迹（Western blot）、透射电子显微镜以及粒径分析对外泌体进行鉴定。将Cal27-exo与NHGFs共培养，检测NHGFs对Cal27-exo的摄取情况，用划痕实验和CCK8检测NHGFs+Cal27-exo的迁移和增殖能力，用实时荧光定量聚合酶链反应检测NHGFs活化相关蛋白金属基质蛋白酶9（MMP-9）、成纤维细胞活化蛋白（FAP）、α平滑肌肌动蛋白（αSMA）和转化生长因子β（TGF-β）的mRNA表达。结果: 超速离心法提取的Cal27-exo中Alix和CD63表达阳性；透射电子显微镜示Cal27-exo呈类圆形双层膜性囊泡；粒径检测示Cal27-exo直径为30~150 nm。共培养后Cal27-exo进入NHGFs内，划痕实验结果示NHGFs+Cal27-exo的迁移能力强于NHGFs，CCK8结果示NHGFs+Cal27-exo的增殖活力强于NHGFs。实时荧光定量聚合酶链反应结果显示，与NHGFs相比，NHGFs+Cal27-exo的MMP-9表达上调，TGF-β、αSMA mRNA表达下调（P<0.05）。结论: NHGFs+Cal27-exo的增殖活性和迁移能力明显增强，相关蛋白的mRNA水平改变。Cal27-exo能活化NHGFs，对肿瘤的侵袭转移有潜在意义。.
Keywords: cell migration; cell proliferation; exosome; fibroblast; oral cancer.