李墨琳 朱亚琼 丁雨菲 易丹 葛乃侨 陈思明 王月香

摘要：目的 探究富血小板血浆源性外泌体（PRP-Exos）对肌腱干/祖细胞（TSPC）增殖及迁移能力的影响。方法 采用聚合沉淀结合超速离心法提取PRP-Exos，采用透射电镜和纳米颗粒跟踪分析技术鉴定其形态、浓度及粒径，采用蛋白免疫印迹法检测外泌体表面标志蛋白及血小板膜糖蛋白的表达水平。提取并培养大鼠TSPC，采用流式细胞术及免疫荧光染色检测TSPC表面标志分子的表达情况。CCK-8法及EdU实验评估PRP-Exos对TSPC增殖情况的影响。细胞划痕实验及Transwell实验评估PRP-Exos对TSPC迁移情况的影响。结果 提取的PRP-Exos呈典型茶托状结构，浓度为4.9×1011个/mL，平均粒径为（132.2±56.8）nm，PRP-Exos表面表达CD9、CD63及CD41。提取的TSPC表达CD44蛋白。PRP-Exos可被TSPC摄取，共培养48 h时，浓度为50、100 μg/mL的PRP-Exos可显著促进TSPC的增殖（P均＜0.001），且两组之间差异无统计学意义（P=0.283）；共培养24 h后，浓度为50 μg/mL的PRP-Exos可显著促进TSPC的迁移（P＜0.001）。结论 体外培养条件下，PRP-Exos对大鼠TSPC的增殖及迁移具有显著促进作用。

关键词：富血小板血浆；外泌体；肌腱干/祖细胞；增殖；迁移；肩袖损伤

中图分类号： R459.9；R686.5  文献标识码： A  文章编号：1000-503X（2024）03-0307-09

DOI：10.3881/j.issn.1000-503X.15890

Effects of Platelet-Rich Plasma-Derived Exosomes on Proliferation and Migration of Tendon Stem/Progenitor Cell

LI Molin1，2，ZHU Yaqiong2，DING Yufei3，YI Dan2，GE Naiqiao1，4，CHEN Siming2，WANG Yuexiang2

1Chinese PLA Medical School，Beijing 100853，China

2Department of Ultrasound，The First Medical Center of Chinese PLA General Hospital，Beijing 100853，China

3Department of Orthopedics，Beijing Friendship Hospital，Capital Medical University，Beijing 100050，China

4Department of Radiology，Armed Police Corps Hospital of Jilin Province，Jilin，Jilin 130000，China

Corresponding author：WANG Yuexiang Tel：010-66935455，E-mail：wangyuexiang1999@sina.com

ABSTRACT：Objective To investigate the effects of platelet-rich plasma-derived exosomes （PRP-Exos） on the proliferation and migration of tendon stem/progenitor cell （TSPC）.Methods PRP-Exos were extracted through the combination of polymer-based precipitation and ultracentrifugation.The morphology，concentration，and particle size of PRP-Exos were identified by transmission electron microscopy and nanoparticle tracking analysis.The expression levels of surface marker proteins on PRP-Exos and platelet membrane glycoproteins were determined by Western blot analysis.Rat TSPC was extracted and cultured，and the expression of surface marker molecules on TSPC was detected using flow cytometry and immunofluorescence staining.The proliferation of TSPC influenced by PRP-Exos was evaluated using CCK-8 assay and EdU assay.The effect of PRP-Exos on the migration of TSPC was evaluated by cell scratch assay and Transwell assay.Results The extracted PRP-Exos exhibit typical saucer-like structures，with a concentration of 4.9×1011 particles/mL，an average particle size of （132.2±56.8） nm，and surface expression of CD9，CD63 and CD41.The extracted TSPC expressed the CD44 protein.PRP-Exos can be taken up by TSPC，and after co-cultured for 48 h，concentrations of 50 and 100 μg/mL of PRP-Exos significantly promoted the proliferation of TSPC （both P<0.001），with no statistical difference between the two concentrations （P=0.283）.Additionally，after co-cultured for 24 h，50 μg/mL of PRP-Exos significantly promoted the migration of TSPC （P<0.001）.Conclusion Under in vitro culture conditions，PRP-Exos significantly promote the proliferation and migration of rat TSPC.

Key words：platelet-rich plasma；exosome；tendon stem/progenitor cell；proliferation；migration；rotator cuff injury

Acta Acad Med Sin，2024，46（3）：307-315

肩袖撕裂是肩关节疼痛最常见的病因，常引发患者的慢性疼痛和功能受限，严重时甚至可能致残［1］。急性肩袖损伤后，及时有效的腱骨界面重塑是良好预后的关键环节。肌腱干/祖细胞（tendon stem/progenitor cell，TSPC）作为重要的内源性干细胞，在肌腱损伤后肌腱细胞的代谢及肌腱功能的维持中起着重要作用，但其增殖及迁移的效率较低［2］。近年研究发现干细胞外泌体可以在一定程度上影响TSPC的增殖及迁移［3］。……