内源性大麻素对缺氧缺糖视网膜神经节细胞损伤的保护作用  

Protection of cannabinoid to retinal ganglion cells against oxygen-glucose deprivation damage

作　　者：沈雨濛 林中乔 刘诗亮 田凯琳 陈媛媛 沈吟 

机构地区：武汉大学人民医院眼科中心,430060

出　　处：《中华实验眼科杂志》2017年第1期10-16,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81270998、81470628);湖北省卫计委青年人才项目(WJ2015Q014);湖北省杰出青年项目(2014CFA019);武汉市科技局晨光计划项目(2016070204010153)

摘　　要：背景急性视网膜缺血缺氧性损伤在眼科较为常见,如青光眼急性发作、视网膜中央动脉阻塞、缺血性视神经病变等,可引起视网膜的缺血缺氧损伤,并可致视网膜神经节细胞（RGCs）的死亡。内源性大麻素（CB）及其受体（CBR）参与中枢神经系统外伤、缺血、炎症及中毒等多种生理病理过程。目的探讨视网膜神经节细胞（RGCs）在缺氧缺糖损伤中内源性CB的作用及意义。方法取6周龄正常C57BL/6J小鼠眼球,制备小鼠视网膜垂直冰冻切片,采用免疫荧光染色法检测和验证CB1R和CB2R在RGCs中的表达。10只C57BL/6J新生鼠浸入75%乙醇消毒后取眼球,在冰上预冷的DMEM培养基中分离视网膜进行RGCs原代培养,采用免疫荧光技术检测培养细胞中RGCs标志物Brn3a的阳性表达并确定培养细胞中CB1R和CB2R的表达。将原代培养14 d的RGCs分为正常对照组和氧糖剥夺（OGD）组,分别用完整培养基+体积分数95%空气+体积分数5%CO2和无糖培养基+体积分数95%N2+4%CO2+体积分数1%O2条件下培养20 h。采用JC-1染色法检测细胞中线粒体结构变化和RGCs的形态变化。各组细胞分别给予1μmol/L CB1R拮抗剂SR141716A、1μmol/L CB2R拮抗剂SR144528以及5μmol/L、10μmol/L CB1R和CB2R激活剂WIN 55212-2,采用MTT法比较各组RGCs存活率。结果正常C57BL/6J小鼠视网膜全层均可见CBR的表达。正常对照组培养的RGCs大小均匀,多呈多角形,细胞间轴突细长并形成网络,细胞中Brn-3a表达阳性;OGD组培养的细胞皱缩或形成碎片,多数细胞轴突消失,Brn-3a表达荧光强度明显减弱。正常对照组RGCs中JC-1染色显示,线粒体的黄色荧光明显强于OGD组,表明OGD组线粒体膜电位明显下降。MTT法检测显示,正常对照组RGCs存活率为（100.00±13.87）%,明显高于OGD组的（89.52±18.16）%,差异均有统计学意义（q=8.065,P=0.008）;SR141716A和SR144528作用后OGD组细胞存活率分别为（116.63±22.21）%和（112Background Acute retinal ischemia anoxic injury is common in eye disorders, such as acute glaucoma,central retinal artery occlusion and ischemic optic neuropathy, etc. This will cause retinal ischemia anoxic injury and induce retinal ganglion ceils （RGCs） death in addition. Endogenous cannabinoid （CB） and its receptors are involved in the central nervous system injury, ischemia, inflammation, and poisoning and other physiological and pathological process. Objective This study was to investigate the effect of CB on RGCs damage induced by oxygen-glucose deprivation （OGD）. Methods The eyeballs were obtained from 6-week-old normal C57BL/6J mice to prepare retinal frozen sectionsand the expression and distribution of cannabinoid receptors （CB1R and CB2R） in RGCs was detected by immunofluorescence staining. The eyeballs of ten newborn C57BL/6J mice （postnatal 0-3 days） were obtained after immersed by 75% alcohol and the retinas were isolated in precooling DMEM for the primary culture of RGCs. The cells were identified by detecting the expression of Brn3a, a marker of RGCs, with immunofluorescence staining. Then the ceils cuhured for 14 days were divided into normal control group （ in complete cuhure medium＋95% air＋5% CO2 ） and OGD group （in glucose-free medium＋95% N2 ＋4% CO2 ＋ 1% 02 ） for 20 hours. The mitochondrial damage and RGCs morphology changed were evaluated by JC-1 staining to observe the mitochondrial membrane potential change. SR141716A （ CB1R antagonist, 1 μmol/L） , SR144528 （ CB2R antagonist, 1 μmol/L） and 5 or 10 μmol/L WIN 55212-2 （ CB1R and CB2R agonist） were added,and the survival rate of RGCs was assayed MTT. Results CBR was positively expressed in various layers of normal mouse retinas. The cells in the normal control group showed uniform size and polygon in shape with the long and thin axons, and the expression of Brn-3a was seen in the cells. However,in the OGD group, cell shrinkage and fragments were found and most of the axons disappeared. The e

关 键 词：大麻素/药物 神经保护剂/药物 大麻素受体/代谢 视网膜神经节细胞 糖/代谢 氧/代谢 C57BL/6J小鼠 氧糖剥夺 

分 类 号：R774.1[医药卫生—眼科]