背景:肌腱是连接肌肉和骨骼的重要致密纤维结构,肌腱干细胞(TDSC)影响其修复和再生。TDSC衍生的外泌体(TDSC-Exos)的作用仍未被探索;因此,本研究旨在探讨TDSC-Exos对肌腱细胞的保护作用。
方法:TDSC和肌腱细胞均来自SD大鼠。流式细胞术检测TDSCs阳性和阴性标志物的表达,并且还检测了多分化能力以鉴定TDSC。Exos使用超速离心从TDSC衍生;此外,转染后,从稳定过表达miR-377-3p的TDSC产生富含微小RNA(miR)-377-3p的Exos,用透射电子显微镜(TEM)鉴定,蛋白质印迹和PKH26染色测定。此外,通过MTT评估肌腱细胞的细胞功能,EdU,transwell,和流式细胞术。使用双荧光素酶报告基因和RNA下拉测定来验证通过Targetscan预测的miR-337-3p和caspase3(CASP3)的结合位点。
结果:Exos(miR-337-3p)被肌腱细胞吸收,并促进了扩散,迁移,和侵袭,并以剂量依赖的方式抑制肌腱细胞的凋亡。生物信息学分析显示CASP3是miR-377-3p的靶标,通过荧光素酶和RNA下拉法进一步验证。此外,过表达的CASP3逆转了Exos(miR-337-3p)对肌腱细胞功能的影响。
结论:我们的研究结果表明,来自miR-337-3p过表达TDSC的Exos可能通过调节CASP3来预防肌腱细胞凋亡。这种新颖的治疗方法有望治疗肌腱损伤,为改善患者预后提供一线希望。
BACKGROUND: Tendons are important dense fibrous structures connecting muscle to bone, and tendon stem cells (TDSCs) affect their repair and regeneration. The role of TDSC-derived exosomes (TDSC-Exos) is still being unexplored; therefore, this study aimed to investigate the protective effect of TDSC-Exos on tenocytes.
METHODS: The TDSCs and tenocytes were all derived from Sprague Dawley (SD) rats. The expression of positive and negative markers of TDSCs were detected by flow cytometry, and the multi-differentiation ability was also detected to identify TDSCs. Exos were derived from TDSCs using ultracentrifugation; furthermore, Exos enriched with microRNA(miR)-377-3p were generated from TDSCs stably overexpressing miR-377-3p after transfection, identified with transmission electron microscopy (TEM), western blot and PKH26 staining assay. Moreover, the cell functions of tenocytes were evaluated by MTT, EdU, transwell, and flow cytometry. Dual luciferase reporter and RNA pull-down assays were used to verify the binding sites of miR-337-3p and caspase3 (CASP3) predicted by Targetscan.
RESULTS: Exos (miR-337-3p) were taken up by tenocytes, and promoted the proliferation, migration, and invasion and suppressed the apoptosis of tenocytes in a dose-dependent manner. Bioinformatics analysis showed that CASP3 was a target of miR-377-3p, which was further verified by luciferase and RNA pull-down assays. Moreover, over-expressed CASP3 reversed the effects of Exos (miR-337-3p) on cell functions of tenocytes.
CONCLUSIONS: Our findings suggest that Exos derived from miR-337-3p over-expressing TDSCs could potentially protect against
tenocyte apoptosis by regulating CASP3. This novel therapeutic approach holds promise for the treatment of tendon injury, offering a glimmer of hope for improved patient outcomes.