一氧化氮(NO)对角膜神经再生的影响作用  

Effect of gas signal molecule nitric oxide on corneal nerve regeneration

作　　者：向征 石赟懿 谭钢[1] XIANG Zheng;SHI Yunyi;TAN Gang(Department of Ophthalmology,the First Affiliated Hospital,University of South China,Hengyang 421005,Hunan Province,China)

机构地区：[1]南华大学附属第一医院眼科,湖南省衡阳市421005

出　　处：《眼科新进展》2022年第10期769-774,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:81100648,82171031);湖南省自然科学基金资助项目(编号:2021JJ30620);湖南省卫生健康委员会科技计划资助项目(编号:20201966)。

摘　　要：目的探讨一氧化氮(NO)对角膜神经再生的影响作用。方法本研究以亚硝酸钠(NaNO_(2))作为外源性NO供体,在细胞实验中以小鼠神经母细胞瘤细胞(Neuro-2a)为研究对象。采用不同浓度的NaNO_(2)处理Neuro-2a细胞并筛选出NO的最佳神经营养浓度。在动物实验中,将30只SD大鼠随机分组,10只作为NC组,其余20只大鼠建立角膜碱烧伤模型,再随机分为PBS组和NO组,每组10只。从碱烧伤当天开始,PBS组给予PBS治疗,NO组给予10.00μmol•L^(-1) NaNO_(2)与PBS混合治疗。用荧光素钠染色后观察并记录大鼠角膜上皮愈合情况,计算角膜上皮愈合率。用CCK-8检测细胞活性,流式细胞术检测细胞凋亡率,免疫荧光法检测细胞神经元标记物的表达。于大鼠角膜碱烧伤处理后7 d取大鼠角膜上皮组织,分别采用实时荧光定量PCR和Western blot法检测每组角膜上皮中神经元标志物βⅢ-微管蛋白和神经生长因子(NGF)、胶质细胞源性神经营养因子(GDNF)、睫状神经营养因子(CNTF)的表达水平。结果10.00μmol•L^(-1) NaNO_(2)处理Neuro-2a细胞24 h后可显著提高细胞活性;使用浓度为0.00μmol•L^(-1)、10.00μmol•L^(-1)、1000.00μmol•L^(-1)的NaNO_(2)处理Neuro-2a细胞24 h后,测得细胞凋亡率分别为18.60%、13.00%、19.48%;与0.00μmol•L^(-1)组相比,10.00μmol•L^(-1)组细胞凋亡率降低(P<0.01)。与0.00μmol•L^(-1)组相比,10.00μmol•L^(-1)组Neuro-2a细胞βⅢ-微管蛋白、MAP2和SMI312三种神经元标志物相对表达量均增加(均为P<0.05)。碱烧伤后1 d、3 d、7 d,与PBS组相比,NO组大鼠角膜上皮愈合率均升高(均为P<0.05)。与NC组相比,PBS组和NO组大鼠角膜组织各神经营养因子mRNA的相对表达量均增高(均为P<0.05)。与PBS组相比,NO组大鼠角膜组织NGF、GDNF、CNTF mRNA的相对表达量均增高(均为P<0.05)。Western blot检测结果显示:碱烧伤后7 d,与NC组相比,PBS组和NO组大鼠角膜组织βⅢ-微管蛋白、NGF、GDNF�Objective To investigate the effect of the gas signal molecule nitric oxide(NO)on corneal nerve regeneration.Methods In this study,sodium nitrite(NaNO_(2))was used as an exogenous NO donor.In cell experiments,mouse neuroblastoma cells(Neuro-2a)were selected and treated with different concentrations of NaNO_(2) to screen out the optimal neurotrophic concentration of NO.In animal experiments,30 SD rats were selected,and 10 of them were randomly divided into the normal control(NC)group.The remaining 20 rats were used to establish corneal alkali burn models and then were randomly divided into the PBS group and the NO group,with 10 rats in each group.From the day of alkali burn,rats in the PBS group were treated with PBS,and rats in the NO group were treated with 10.00μmol•L^(-1) NaNO_(2) mixed with PBS.After fluorescein sodium staining,the healing of corneal epitheliums in rats was observed and recorded,and its rate was calculated.The cell viability was detected by CCK-8,the cell apoptosis by flow cytometry,and the expression of neuronal markers by immunofluorescence.Rat corneal epithelial tissues were collected seven days after alkali burn,and the expression levels ofβⅢ-tubulin,nerve growth factor(NGF),glial cell-derived neurotrophic factor(GDNF),and ciliary neurotrophic factor(CNTF)in them were measured by real-time quantitative polymerase chain reaction and Western blot.Results The viability of Neuro-2a cells treated with 10.00μmol•L^(-1) NaNO_(2) for 24 h was significantly increased.The apoptosis of Neuro-2a cells treated with 0.00μmol•L^(-1),10.00μmol•L^(-1),and 1000.00μmol•L^(-1) NaNO_(2) for 24 h was 18.60%,13.00%,and 19.48%,respectively.Compared with the 0.00μmol•L^(-1) group,the apoptosis rate in the 10.00μmol•L^(-1) group decreased(P<0.01),while the relative expression levels ofβⅢ-tubulin,microtubule-associated protein 2 and SMI 312 increased(all P<0.05).Compared with the PBS group,the corneal epithelial healing rate in the NO group significantly improved one,three and seven days

关 键 词：角膜碱烧伤 一氧化氮 角膜神经再生 神经生长因子 

分 类 号：R772.2[医药卫生—眼科]