PLGA微球载NGF联合超声对视神经节细胞的保护作用

Protective effect of NGF loaded PLGA microspheres combined with ultrasonic irradiation on the rat retinal ganglion cells following the optic nerve injury

目的:探讨乳酸-羟基乙酸共聚物(ploy lactic-co-glycolic acid,PLGA)微球联合神经生长因子(nerve growth factor,NGF)对视神经损伤大鼠视神经节细胞(retinal ganglion cells,RGCs)的保护作用。方法:PLGA为原料制备包载有NGF的PLGA微球。检测载药微球的载药量和包封率,观察其形态特点测量粒径分布。将Sprague-Dawley(SD)雄性成年大鼠69只随机分为5个组,正常组(A组)和假手术组(B组)各9只大鼠,生理盐水注射组(C组)、神经生长因子注射组(D组)、PLGA微球联合NGF注射组(E组)各17只大鼠,B^E组大鼠均行超声辐照处理。各组分别于0.5、1、3、7 d取样行视网膜切片免疫组化染色和和苏木精-伊红染色(hematoxylin-eosin staining,HE),观察视网膜上葡萄糖钙调蛋白(glucose regulated rrotein 78 k D,GRP78)表达和组织病理的变化,并采用荧光金(fluorogold,FG)逆行标记视神经节细胞于视神经钳夹损伤造模后第7 d取样计数各组RGCs数量。结果:PLGA载药微球包封率68.4%,载药量0.032 3%,体外7 d的累计释放率82.6%,微球粒径(3.17±0.6)μm。FG逆行标记RGCs计数5组间差异有统计学意义(F=65.858,P=0.000),A组与B组间差异无统计学意义(P=0.862>0.05),其它各组间差异均有统计学意义(P<0.05)。免疫组化染色A组、B组未见阳性细胞,RGCs的GRP78阳性细胞率显示,C^E组不同时间各组间差异有统计学意义(F组别=184.330,P组别=0.000),各组在各时间点间差异亦有统计学意义(F时间=21.472,P时间=0.000)。HE染色提示第7天时E组视网膜水肿较C、D组减轻。结论:NGF-PLGA微球联合超声治疗视神经损伤大鼠能提高对RGCs的保护作用。

Objective： To observe the protective effect of nerve growth factor （NGF） loaded ploy lactic-co-glycolic acid （PLGA） microspheres on retinal ganglion cells （RGCs） following the optic nerve injury. Methods：The NGF-PLGA microspheres were made using PLGA and the properties of microspheres were tested. Totally 69 SD adult male rats were randomly divided into 5 groups：normal control group （group A）, sham operated group （group B）, physiological saline injection group （group C）, N GF injection group （group D） and NGF-PLGA injection group（group E）. Nine rats in groups A and B, 17 rats in groups C,D and E. All rats in groups B-E underwent ultrasound irradiation. Samples of retinal section were collected for immunohistochemistry staining and HE staining to observe the performance of glucose regulated protein 78 kD（GRP78） and the pathological morphology change of retina of each group at 12 h and on the 1st d, 3rd d, 7th d. Number of RGCs retrograde labeled by fluorogold （FG） before the operation was checked on the 7th d. Results ：The mean size of microsheres was （3.17 ± 0.60） μm. The encapsulation efficiency of NGF-PLGA microspheres was 68.4%,the loading rate was 0.0323% and the cumulative rate of drug-release on the 7th d was 82.6%.There were significant differences among 5 groups in RGCs count（F=65.858,P=-0.000）.There was no difference in RGCs count between group A and B（P=-0.862〉 0.05）, but apparent differences among other groups were observed（P〈0.05）. Imunohistochemistry staining showed no positive cell in groups A or B. There were significant differences in GRP78 positive rate of RGCs among groups C-E at different time points（Fgroup= 184.330,Pgroup=0.000） and among each group at different time points（Ftime=21.472,Ptime=0.000）. HE staining showed the retinal edema of group E was reduced on the 7th d compared with that of group C and D. Conclusion：NGF loaded PLGA microspheres combined with ultrasonic irradiation can improve the protective effect on RGCs after the optic nerve injury.