基于促红细胞生成素-葡聚糖微粒的 PLGA/PLA 微球对 RGCs 的影响。
Effects of erythropoietin-dextran microparticle-based PLGA/PLA microspheres on RGCs.
机构信息
Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China.
出版信息
Invest Ophthalmol Vis Sci. 2012 Sep 7;53(10):6025-34. doi: 10.1167/iovs.12-9898.
PURPOSE
We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time.
METHODS
EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate).
RESULTS
The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush).
CONCLUSIONS
A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.
目的
我们探索了载红细胞生成素（EPO）葡聚糖微粒的聚（DL-乳酸-共-乙醇酸）/聚（DL-乳酸）（PLGA/PLA）微球（EPO-葡聚糖 PLGA/PLA 微球）对视神经挤压大鼠视网膜神经节细胞（RGCs）的长期神经保护作用。
方法
首先通过一种新型的固-油-水（S/O/W）技术制备 EPO-葡聚糖 PLGA/PLA 微球。然后，评估体外 EPO 释放曲线。随后，在视网膜外植体上研究 EPO 从 EPO-葡聚糖 PLGA/PLA 微球中释放的生物活性作用。最后，通过 TUNEL 染色评估视神经挤压大鼠中 EPO-葡聚糖 PLGA/PLA 微球对 RGCs 的神经保护作用，用于凋亡 RGCs。通过免疫组织化学染色研究视网膜中胶质纤维酸性蛋白（GFAP）的表达水平。使用 DiI 荧光示踪剂（1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐）通过 DiI 逆行标记观察存活的 RGCs。
结果
结果表明，PLGA/PLA 微球中 EPO 的持续释放至少可以持续 60 天。微球中释放的 EPO 在视网膜外植体上表现出与 EPO 蛋白溶液同样有效的神经再生作用（神经突密度的 P 值为 0.2554，神经突长度的 P 值为 0.1004）。TUNEL 染色显示，与对照组（未处理组）相比，EPO-葡聚糖 PLGA/PLA 微球显著减少了 RGCs 的死亡（视神经挤压后 5 天和 1 周时 P <0.01，视神经挤压后 2 周时 P <0.05）。视神经挤压后 2 周，EPO-葡聚糖 PLGA/PLA 微球给药大鼠视网膜中 GFAP 的表达明显减少。DiI 逆行标记显示，单次玻璃体内注射 EPO-葡聚糖 PLGA/PLA 微球显著促进了 RGCs 的存活（视神经挤压后 4 周和 8 周时 P <0.01）。
结论
单次玻璃体内注射 EPO-葡聚糖 PLGA/PLA 微球对视神经挤压大鼠的 RGCs 似乎具有长期的保护作用。PLGA/PLA 微球可能是一种可行的蛋白质递药系统，例如 EPO，可用于玻璃体内注射治疗视网膜变性疾病。
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