BDNF过表达的外泌体对缺血性视网膜损伤的保护作用及机制研究
收稿日期: 2026-03-17
修回日期: 2026-05-07
录用日期: 2026-08-13
网络出版日期: 2026-08-17
基金资助
国家自然科学基金项目（81270146）；首都医科大学校自然培育基金项目（PYZ18115）
Protective Effects and Mechanism of Exosomes Overexpressing BDNF on Ischemic Retinal Injury
Received date: 2026-03-17
Revised date: 2026-05-07
Accepted date: 2026-08-13
Online published: 2026-08-17
目的：探讨过表达脑源性神经营养因子（BDNF）的人293T细胞来源外泌体（293T-Exo）对视网膜缺血损伤的保护作用及潜在的内吞机制。方法：采用超速离心结合试剂盒法从BDNF过表达的293T细胞培养上清中分离外泌体，通过透射电镜、纳米颗粒追踪分析与Western blot进行鉴定。将293T-Exo与R28细胞共培养，采用荧光标记技术观察细胞摄取情况，并通过RGD肽、肝素及甲基-β-环糊精（MBCD）干预实验探讨其内吞机制。建立R28细胞氧-葡萄糖剥夺（OGD）模型模拟体外缺血损伤，采用EdU掺入法和乳酸脱氢酶释放法评估293T-Exo对细胞增殖与死亡的影响。构建SD大鼠视网膜缺血再灌注模型，玻璃体腔注射293T-Exo后，通过TUNEL染色检测视网膜组织凋亡，免疫荧光染色观察外泌体在视网膜神经节细胞（RGCs）中的定位。结果：成功分离得到直径约为144.2 nm、表达CD9、CD63、CD81和HSP70α的典型外泌体颗粒。293T-Exo可被R28细胞以剂量和温度依赖的方式摄取；该过程可被RGD肽与MBCD显著抑制，且外泌体与Caveolin-1存在共定位，提示其通过整合素介导的小窝蛋白依赖性内吞途径进入细胞。与对照组相比，293T-Exo处理可显著增强OGD条件下R28细胞的增殖能力并降低细胞死亡率（P<0.05）。体内实验显示，玻璃体注射293T-Exo可显著减少缺血视网膜各层（尤其是RGC层）的TUNEL阳性细胞数（P<0.05），且外泌体主要被RGCs摄取。结论：过表达BDNF的293T-Exo可通过整合素介导的小窝蛋白依赖性内吞途径被视网膜细胞有效摄取，并在体内外缺血模型中发挥显著的神经保护作用，提示其作为视网膜缺血性疾病无细胞治疗策略的潜在应用价值。
关键词： ; 视网膜缺血；外泌体；脑源性神经营养因子；内吞作用；凋亡
闫博婧
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BDNF过表达的外泌体对缺血性视网膜损伤的保护作用及机制研究
Objective： To investigate the protective effect of exosomes derived from human 293T cells overexpressing brain-derived neurotrophic factor （BDNF） （293T-Exo） on retinal ischemic injury and its potential endocytic mechanism.Methods： Exosomes were isolated from the culture supernatant of BDNF-overexpressing 293T cells using ultracentrifugation combined with a kit method， and identified by transmission electron microscopy （TEM）， nanoparticle tracking analysis （NTA）， and Western blot. 293T-Exo was co-cultured with R28 cells， and cellular uptake was observed using fluorescence labeling. The endocytic mechanism was explored via intervention experiments using RGD peptide， heparin， and methyl-β-cyclodextrin （MBCD）. An oxygen-glucose deprivation （OGD） model in R28 cells was established to simulate ischemic injury in vitro. EdU incorporation assay and lactate dehydrogenase （LDH） release assay were used to evaluate the effects of 293T-Exo on cell proliferation and death. A retinal ischemia-reperfusion model was constructed in SD rats. After intravitreal injection of 293T-Exo， retinal apoptosis was detected by TUNEL staining， and the localization of exosomes in retinal ganglion cells （RGCs） was observed by immunofluorescence staining.Results： Typical exosome particles with a diameter of approximately 144.2 nm expressing CD9， CD63， CD81， and HSP70α were successfully isolated. 293T-Exo was taken up by R28 cells in a dose- and temperature-dependent manner. This process was significantly inhibited by RGD peptide and MBCD， and co-localization with Caveolin-1 was observed， suggesting an integrin-mediated caveolin-dependent endocytic pathway. Compared with the control group， 293T-Exo treatment significantly enhanced the proliferative capacity of R28 cells under OGD conditions and reduced cell mortality （P<0.05）. In vivo experiments showed that intravitreal injection of 293T-Exo significantly reduced the number of TUNEL-positive cells in all layers of the ischemic retina， especially in the RGC layer （P<0.05）， and the exosomes were mainly taken up by RGCs.Conclusion： 293T-Exo of overexpressing BDNF can be effectively taken up by retinal cells via an integrin-mediated caveolin-dependent endocytic pathway and exert significant neuroprotective effects in both in vitro and in vivo ischemic models， suggesting their potential application value as a cell-free therapeutic strategy for ischemic retinal diseases.
Key words： ; Retinal ischemia； ; Exosomes； ; Brain-derived neurotrophic factor； ; Endocytosis； ; Apoptosis
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