Objective: To investigate the protective effect of bone marrow mesenchymal stem cells (BMSCs)-derived exosomesagainst testicular ischemia-reperfusion injury (IRI) in rats.
Methods: Rat BMSCs were isolated, cultured and identified in theprimary culture. The exosomes were extracted from the BMSCs and characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Twenty-four healthy male SD rats were randomly divided into shamoperation group, testicular IRI with saline treatment group and IRI with exosome treatment group. The contralateral testes ofthe rats were collected for pathological observation, aseessment of superoxide dismutase (SOD) and malondialdehyde (MDA), and detection of HMGB1, caspases-3 and cleaved caspase-3 expressions using Western blotting.
Results: We successfullyobtained exosomes from rat BMSCs. Testicular IRI significantly impaired testicular spermatogenesis, which was markedlyimproved by treatment with the exosomes (P < 0.05). Testicular IRI also caused significant increase in the protein expression ofHMGB1, caspase-3 and cleaved caspase-3 in the testicular tissue, and treatment with the exosomes obviously amelioratedthese changes (P < 0.05).
Conclusions: BMSCs-derived exosomes protects against testicular IRI due to the anti-oxidant, antiinflammatory and anti-apoptosis activities of the exosomes.
目的: 探讨大鼠骨髓间充质干细胞（BMSCs）来源的外泌体对睾丸缺血再灌注损伤的保护作用。
方法: 采用原代培养方法分离、培养、并鉴定大鼠BMSCs。超高速离心法提取BMSCs来源的外泌体，并采用纳米颗粒跟踪分析仪（NTA）分析其粒径大小，透射电子显微镜观察其形态，蛋白免疫印迹法检测其典型标志蛋白质鉴定提取的外泌体。构建雄性SD大鼠睾丸缺血再灌注损伤（IRI）模型，24只健康雄性SD大鼠随机分为3组；A组：假手术组（Sham组），B组：生理盐水处理组（I/R+NS），C组：BMSCs来源外泌体（100 μg/mL）处理组（I/R+BMSCs-exo）。最后取各组大鼠扭转侧（左侧）睾丸并用光学显微镜检测睾丸组织病理学结构改变以及对生精结构进行组织评分，采用生物化学的方法检测各组睾丸组织中超氧化物歧化酶的活性，丙二醛的含量，蛋白免疫印迹法检测高迁移率族蛋B1（HMGB1），含半胱氨酸的天冬氨酸蛋白水解酶3（caspases-3）和剪切型caspase-3的表达。
结果: 成功从大鼠骨髓中原代分离并培养BMSCs，并成功提取了BMSCs分泌的外泌体。动物模型中，与睾丸生精结构正常的A组（Sham）相比，B组（I/R+NS）的睾丸生精结构明显有不同程度受损，而C处理组（I/R+BMSCs-exo）中有一定的改善（P < 0.05）。睾丸组织生化指标检测中，B组（I/R+NS）组织中的MDA的含量较A组（Sham）明显升高，而SOD的活性与A组（Sham）相比则降低（P < 0.01），而在经外泌体处理之后的C组（I/R+BMSCs-exo）中，与生理盐水处理组B组相比，组织中MDA的含量有一定程度降低而SOD的水平则上调（P < 0.05）。睾丸组织蛋白免疫印迹上，与A组（Sham）相比，B组（I/R+NS）的HMGB1，caspase-3以及剪切型caspase-3明显升高，而C组（IR+BMSCs-exo）的HMGB1，caspase-3和剪切型caspase-3一定程度的降低（P < 0.05）。
结论: BMSCs来源的外泌体对睾丸缺血再灌注损伤有抗氧化，抗炎症和抗凋亡的保护作用。
Keywords: antioxidant; apoptosis; bone marrow mesenchymal stem cells; exosomes; inflammation; ischemia-reperfusion injury; testis.