NEP1-40对视网膜缺血-再灌注损伤过程中视网膜细胞的保护作用
NEP1-40대시망막결혈-재관주손상과정중시망막세포적보호작용
Protection of NEP1-40 on retinal cells following retinal ischemia reperfusion injury
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2014年 3期 220-225 ,共6页

    刘索新%鞠学红%于树娜%王浩

    류색신%국학홍%우수나%왕호

    NEP1-40%视网膜%缺血-再灌注损伤%Nogo-A%凋亡 NEP1-40%視網膜%缺血-再灌註損傷%Nogo-A%凋亡
    NEP1-40%시망막%결혈-재관주손상%Nogo-A%조망
    NEP1-40%Retina%Ischemia reperfusion injury%Nogo-A%Apoptosis
    背景 视网膜缺血-再灌注损伤(RIRI)可导致细胞凋亡,凋亡是一个复杂的过程,与许多基因有关,探讨药物对RIRI后视细胞凋亡的保护作用具有重要意义. 目的 探讨Nogo-A在大鼠RIRI模型中的表达及其与细胞凋亡的关系,研究Nogo受体竞争性拮抗剂NEP1-40对视网膜细胞的保护作用. 方法 将78只SD大鼠按随机数字表法随机分为正常组、RIRI组和NEP1-40组,RIRI组及NEP1-40组大鼠分别用前房升高眼压的方法升高眼压至大鼠视网膜苍白,持续60 min,然后恢复眼压至视网膜色泽恢复,制作RIRI大鼠模型.术后6h、12 h及1、2、3、7d用过量的水合氯醛处死大鼠,制备视网膜标本.透射电子显微镜下观察各时间点各组大鼠视网膜的超微结构,采用TUNEL法检测视网膜细胞的凋亡情况,并计算细胞凋亡指数(AI).采用免疫组织化学法检测视网膜组织中Nogo-A蛋白的表达,并采用逆转录PCR(RT-PCR)法半定量检测Nogo-A mRNA在视网膜中的表达. 结果 正常组大鼠视网膜各层结构正常；RIRI组大鼠视网膜再灌注12h后可见少量视网膜细胞器的线粒体嵴异常及空泡化,再灌注后1 ～2d可见凋亡小体；NEP1-40组视网膜的视细胞外节膜盘略疏松,部分线粒体嵴短小,空泡化,未见凋亡小体,视网膜细胞超微结构损害明显轻于RIRI组.TUNEL检测显示细胞凋亡出现于再灌注后6h,并逐渐递增,再灌注后1d凋亡细胞数目达高峰,2d后开始下降,但再灌注后3d仍可见TUNEL阳性细胞.NEP1-40组各时间点间凋亡细胞数目较RIRI组明显减少.正常组、RIRI组和NEP1-40组大鼠在不同时间点细胞AI的差异均有统计学意义(F分组=100.850,P=0.000;F时间=34.309,P=0.000),其中RIRI组和NEP1-40组大鼠视网膜细胞AI明显高于正常组,而NEP1-40组AI明显低于RIRI组,差异均有统计学意义(均P＜0.05).Nogo-A蛋白及其mRNA主要表达于RIRI后的视网膜,再灌注后6h大鼠视网膜中Nogo-A蛋白及其mRNA均开始增加,至再灌注id时表达达高峰,然后逐渐减弱,但再灌注后7d仍有表达.3个组大鼠不同时间点视网膜中Nogo-A蛋白的表达差异有统计学意义(F分组=164.139,P=0.000; F时间=21.772,P=0.000),Nogo-A mRNA的表达差异有统计学意义(F分组=93.889,P=0.000;F时间=6.349,P=0.000),NEP 1-40组和RIRI组大鼠视网膜中Nogo-A蛋白及其mRNA的表达均明显高于正常组,而NEP1-40组大鼠视网膜中Nogo-A蛋白及其mRNA在各时间点的表达均低于RIRI组,差异均有统计学意义(P＜0.05). 结论 Nogo-A可促进RIRI后视网膜细胞的凋亡,NEP1-40能抑制Nogo-A的表达,从而减少视网膜细胞的凋亡,对RIRI后的视网膜细胞有保护作用.
    배경 시망막결혈-재관주손상(RIRI)가도치세포조망,조망시일개복잡적과정,여허다기인유관,탐토약물대RIRI후시세포조망적보호작용구유중요의의. 목적 탐토Nogo-A재대서RIRI모형중적표체급기여세포조망적관계,연구Nogo수체경쟁성길항제NEP1-40대시망막세포적보호작용. 방법 장78지SD대서안수궤수자표법수궤분위정상조、RIRI조화NEP1-40조,RIRI조급NEP1-40조대서분별용전방승고안압적방법승고안압지대서시망막창백,지속60 min,연후회복안압지시망막색택회복,제작RIRI대서모형.술후6h、12 h급1、2、3、7d용과량적수합록철처사대서,제비시망막표본.투사전자현미경하관찰각시간점각조대서시망막적초미결구,채용TUNEL법검측시망막세포적조망정황,병계산세포조망지수(AI).채용면역조직화학법검측시망막조직중Nogo-A단백적표체,병채용역전록PCR(RT-PCR)법반정량검측Nogo-A mRNA재시망막중적표체. 결과 정상조대서시망막각층결구정상；RIRI조대서시망막재관주12h후가견소량시망막세포기적선립체척이상급공포화,재관주후1 ～2d가견조망소체；NEP1-40조시망막적시세포외절막반략소송,부분선립체척단소,공포화,미견조망소체,시망막세포초미결구손해명현경우RIRI조.TUNEL검측현시세포조망출현우재관주후6h,병축점체증,재관주후1d조망세포수목체고봉,2d후개시하강,단재관주후3d잉가견TUNEL양성세포.NEP1-40조각시간점간조망세포수목교RIRI조명현감소.정상조、RIRI조화NEP1-40조대서재불동시간점세포AI적차이균유통계학의의(F분조=100.850,P=0.000;F시간=34.309,P=0.000),기중RIRI조화NEP1-40조대서시망막세포AI명현고우정상조,이NEP1-40조AI명현저우RIRI조,차이균유통계학의의(균P＜0.05).Nogo-A단백급기mRNA주요표체우RIRI후적시망막,재관주후6h대서시망막중Nogo-A단백급기mRNA균개시증가,지재관주id시표체체고봉,연후축점감약,단재관주후7d잉유표체.3개조대서불동시간점시망막중Nogo-A단백적표체차이유통계학의의(F분조=164.139,P=0.000; F시간=21.772,P=0.000),Nogo-A mRNA적표체차이유통계학의의(F분조=93.889,P=0.000;F시간=6.349,P=0.000),NEP 1-40조화RIRI조대서시망막중Nogo-A단백급기mRNA적표체균명현고우정상조,이NEP1-40조대서시망막중Nogo-A단백급기mRNA재각시간점적표체균저우RIRI조,차이균유통계학의의(P＜0.05). 결론 Nogo-A가촉진RIRI후시망막세포적조망,NEP1-40능억제Nogo-A적표체,종이감소시망막세포적조망,대RIRI후적시망막세포유보호작용.
    Background The retina ischemia reperfusion injury (RIRI) can lead to apoptosis,which is associated with many genes.It is very significant to explore the protection of drugs on RIRI-induced apoptosis.Objective This study was to explore the relationship between the expression of Nogo-A and the cell apoptosis as well as the therapeutic effect of NEP1-40 in RIRI retina.Methods The device of raising intraocular pressure (IOP)was used to elevate the IOP for 60 minutes and then restore the IOP to normal for the establishment of RIRI models.Seventy-eight SD rats were randomized to the normal group,RIRI group and NEP1-40 group.PBS of 5 ml/(kg · d)was injected intraperitoneally in the rats of the RIRI group,and NEP1-40 of 8 mg/(L · kg) was used in the same way in the NEP1-40 group.The rats were sacrificed in overdose anesthesia at 6 hours,12 hours and 1 day,2,3,7 days after RIRI to prepare the retinal specimens.The ultrastructure of rat retinas was examined under the transmission electron microscope.Cell apoptosis was assessed by the TUNEL method,and the apoptotic index (AI) was calculated.The expressions of Nogo-A protein and mRNA in rat retinas were detected by immunohistochemistry and semiquantitative reverse transcription PCR (RT-PCR).Results The ultrastructure of retinal cells were normal in the normal group.Retinal cell organelle dissolution,mitochondria swelling,cavitation,chromatin edge heterochromatin and apoptotic body were seen in the rats of the RIRI group from 12 hours through 7 days after injection.However,only the slight loose of outer membranous disk,inner and outer nuclear layer and retinal ganglion cells,the nucleus gap broadening,shortness of some mitochondrial cristae in the NEP1-40 group.TUNEL-positive cells appeared 6 hours after RIRI and reached peak in 1 day,and gradually declined after that in the RIRI group.A similar pattern was seen in the rats of the NEP1-40 group with a more mild manifestation.Significant differences were seen in the AI values among the different groups at various time points (Fgroup =100.850,P =0.000 ; Ftime =34.309,P =0.000),and the AI values were significantly higher in the RIRI group and NEP1-40 group compared with normal group;while the AI values in the NEP1-40 group was lower than those of the RIRI group (all at P＜0.05).The expressions of Nogo-A protein and mRNA showed a coincident pattern with apoptosis procedure,with a gradually elevated level from 6 hours through 7 days after RIRI and a peak in 2 days,and those in the NEP1-40 group were weaker in comparison with the RIRI group in various time points.Significant differences were detected in the expression of Nogo-A protein and the expression of Nogo-A mRNA among different groups and various time points(protein:Fgroup =164.139,P =0.000;Ftime =21.772,P =0.000.mRNA:Fgroup =93.889,P =0.000 ; Ftime =6.349,P =0.000).Conclusions Nogo-A probably plays an important role in RIRI.NEP1-40 can protect retinal cells from apoptosis following RIRI through down-regulating the expression of Nogo-A.
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