[1]李欣蒙,高洪莲,刘奇奇,等.雷帕霉素对豚鼠形觉剥夺性近视的干预作用及相关机制研究[J].眼科新进展,2021,41(3):212-217.[doi:10.13389/j.cnki.rao.2021.0044]
　LI Xinmeng,GAO Honglian,LIU Qiqi,et al.Effects of rapamycin on the form-deprivation myopia in guinea pigs and its related mechanisms[J].Recent Advances in Ophthalmology,2021,41(3):212-217.[doi:10.13389/j.cnki.rao.2021.0044]
点击复制

雷帕霉素对豚鼠形觉剥夺性近视的干预作用及相关机制研究/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年3期
页码:
212-217
栏目:
实验研究
出版日期:
2021-03-05

文章信息/Info

Title:
Effects of rapamycin on the form-deprivation myopia in guinea pigs and its related mechanisms
作者:
李欣蒙; 高洪莲; 刘奇奇; 于睿; 张磊
256600　山东省滨州市，滨州医学院附属医院眼科（李欣蒙，刘奇奇，于睿，张磊）；256600　山东省滨州市，滨州医学院附属医院临床医学实验室（高洪莲）
Author(s):
LI Xinmeng1; GAO Honglian2; LIU Qiqi1; YU Rui1; ZHANG Lei1
1.Department of Ophthalmology,the Affiliated Hospital of Binzhou Medical University,Binzhou 256600,Shandong Province,China
2.Clinical Medical Laboratory of Affiliated Hospital of Binzhou Medical University,Binzhou 256600,Shandong Province,China
关键词:
形觉剥夺性近视; 雷帕霉素; 雷帕霉素靶蛋白; 基质金属蛋白酶-2; α-平滑肌肌动蛋白; 转化生长因子β1; mTOR信号通路
Keywords:
form-deprivation myopia;  rapamycin;  rapamycin targeted proteins;  matrix metalloproteinase-2;  α-smooth muscle actin;  transforming growth factor β1;  mTOR signal pathway
分类号:
R778.1
DOI:
10.13389/j.cnki.rao.2021.0044
文献标志码:
A
摘要:
目的　探讨球结膜下注射雷帕霉素对豚鼠形觉剥夺性近视（FDM）的干预作用及相关机制。方法　选取2～3周龄豚鼠80只（80眼），雌雄不限，随机分为4组：空白对照组、FDM组、雷帕霉素组、FDM+雷帕霉素组；空白对照组不做任何处理；FDM组单纯缝合豚鼠右眼眼睑；雷帕霉素组于实验第1天、第7天豚鼠右眼球结膜下注射雷帕霉素50 μg；FDM+雷帕霉素组于实验第1天、第7天豚鼠右眼球结膜下注射雷帕霉素50 μg后再缝合右眼眼睑。记录各组豚鼠实验前及实验第7天、第14天的眼轴长度、屈光度；并取各组豚鼠视网膜行过碘酸-雪夫染色（PAS染色）及透射电镜检查，观察各组豚鼠视网膜形态变化；取各组豚鼠巩膜行RT-PCR检测巩膜组织中mTOR、基质金属蛋白酶-2（MMP-2）、α-平滑肌肌动蛋白（α-SMA）及转化生长因子β1（TGF-β1）的mRNA表达；利用Western blot 检测各组豚鼠巩膜组织中AKT、p-AKT、mTOR、p-mTOR的蛋白表达。结果　FDM组与空白对照组相比，实验第7天、第14天豚鼠的右眼眼轴长度、屈光度增加，形成了相对近视；FDM组巩膜中MMP-2、α-SMA 的mRNA相对表达量与空白对照组相比均增加，TGF-β1 mRNA相对表达量减少，差异均具有统计学意义（均为P<0.05）；与空白对照组相比，FDM组豚鼠巩膜中mTOR mRNA和AKT、P-AKT、mTOR、p-mTOR蛋白的相对表达量变化不大，差异均无统计学意义（均为P>0.05），但视网膜变薄，各层结构排列紊乱、疏松。雷帕霉素组与空白对照组相比，实验第7天、第14天豚鼠右眼眼轴长度、屈光度差异均无统计学意义（均为P>0.05），且视网膜形态无明显变化。FDM+雷帕霉素组与FDM组相比，实验第7天豚鼠右眼眼轴长度、屈光度差异均无统计学意义（均为P>0.05），实验第14天右眼眼轴长度和屈光度均减小，差异均有统计学意义（均为P<0.05），TGF-β1 mRNA的相对表达量增多，MMP-2、α-SMA、mTOR 的mRNA和AKT、p-AKT、mTOR、p-mTOR的蛋白相对表达量均减少，差异均有统计学意义（均为P<0.05）。结论　雷帕霉素球结膜下注射可延缓豚鼠FDM的形成，可能是通过抑制PI3K/AKT/mTOR信号通路、调节巩膜中的MMP-2、TGF-β1、α-SMA的表达而发挥作用的，且对正常豚鼠视网膜结构没有影响。
Abstract:
Objective　To investigate the effect of subconjunctival injection of rapamycin on the form-deprivation myopia (FDM) of guinea pigs and its related mechanisms.Methods　Together 80 healthy guinea pigs (80 eyes), male or female, aged 2-3 weeks, were randomly divided into 4 groups: blank control group, FDM group, rapamycin group, and FDM+ rapamycin group. Guinea pigs in the blank control group left untreated; in the FDM group, the right eyelids of pigs were only sutured; 1 day and 7 days of the experiment, 50 μg rapamycin was injected subconjunctival in the rapamycin group while the right eyelids were sutured after subconjunctival injection in the FDM+ rapamycin group. The axial length and diopter of each group were recorded before and 7 days and 14 days of the experiment, and the eyeballs were taken for periodate Schiff’s staining (PAS staining) and retinal transmission electron microscope to observe the changes of retinal morphology. The sclera of each group of guinea pigs was taken for RT-PCR to measure mRNA expression of scleral tissue mTOR, matrix metalloproteinase-2 (MMP-2), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1); Western blot was used to detect the protein expression of AKT, p-AKT, mTOR, and p-mTOR in the scleral tissue of guinea pigs in each group.Results　Compared with the blank control group, the right eyes of axial length and diopter in the FDM group increased on day 7 and 14 of the experiment, resulting in relative myopia. The relative expression of MMP-2 and α-SMA mRNA increased while the relative expression of TGF-β1mRNA decreased, and the differences were statistically significant (all P<0.05). Compared with the blank control group, there was no significant difference in the relative expression of mTOR mRNA and AKT, p-AKT, mTOR and p-mTOR protein in the sclera of guinea pigs in the FDM group (all P＞0.05), and the retina became thinner and the structure of each layer becomes disordered and loose. There was no significant difference in axial length and diopter of right eye between rapamycin group and blank control group on day 7 and 14 of the experiment (all P>0.05), and there was no significant change in retinal morphology. There was no significant difference in right eye axial length and diopter between FDM+rapamycin group and FDM group on day 7 of the experiment (both P>0.05), while on day 14 of the experiment, the axial length and diopter of the right eye decreased, and the differences were statistically significant (all P<0.05). The relative expression of TGF-β1 mRNA increased, while the relative expression of MMP-2, α-SMA and mTOR mRNA and AKT, p-AKT, mTOR and p-mTOR protein decreased, and the difference was statistically significant (all P<0.05).Conclusion　Subconjunctival injection of rapamycin can delay the formation of FDM in guinea pigs, which may play a role in inhibiting PI3K/AKT/mTOR signal pathway and regulating the expression of MMP-2, TGF- β1, α-SMA and mTOR in the sclera, and it has no effect on the normal retinal structure.

参考文献/References:

［1］　BOURNE R R,STEVENS G A,WHITE R A,SMITH J L,FLAXMAN S R,PRICE H,et al.Causes of vision loss worldwide,1990-2010:a systematic analysis［J］.Lancet Glob Health,2013,1(6):e339-e349.
［2］　WONG T Y,FERRIRA A,HUGHES R,CARTER G,MITCHELL P.Epidemiology and disease burden of pathologic myopia and myopic choroidal neovascularization:an evidence-based systematic review［J］.Am J Ophthalmol,2014,157(1):9-25.
［3］　JOACHIMSEN L,BOHRINGER D,GROSS N J,REICH M,STIFTER J,REINHARD T,et al.A pilot study on the efficacy and safety of 0.01% atropine in german school children with progressive myopia［J］.Ophthalmol Ther,2019,8(3):427-433.
［4］　SACCHI M,SERAFINO M,VILLANI E,TAGLIABUE E,LUCCARELLI S,BONSIGNORE F,et al.Efficacy of atropine 0.01% for the treatment of childhood myopia in European patients［J］.Acta Ophthalmol,2019,97(8):e1136-e1140.
［5］　WU P C,CHUANG M N,CHOI J,CHEN H,WU G.Update in myopia and treatment strategy of atropine use in myopia control［J］.Eye,2019,33(1):3-13.
［6］　龙琴,褚仁远.巩膜细胞外基质及基质金属蛋白酶在近视发展中作用的研究进展［J］.中华眼科杂志,2005,41(11):1047-1049．
LONG Q,CHU R Y.Scleral extracellular matrix and matrix metalloproteinases play an important role in myopia ［J］.Chin J Ophthalmol,2005，41(11)：1047-1049．
［7］　张兰兰,刘琼,于健,唐晓娟,徐静.转化生长因子-β(TGF-β)影响豚鼠巩膜成纤维细胞增殖和α-平滑肌肌动蛋白(α-SMA)表达的研究［J］.眼科新进展,2016,36(11):1011-1015.
ZHANG L L,LIU Q,YU J,TANG X J,XU J.Effects of transforming growth factor-β (TGF-β) on sclera fibroblast proliferation and α-smooth muscle actin (α-SMA) expression in guinea pigs ［J］.Rec Adv Ophthalmol,2016,36(11):1011-1015.
［8］　BERGMEOER V,ETICH J,PITZLER L,FRIE C,KOCH M,FISCHER M,et al.Identification of a myofibroblast-specific expression signature in skin wounds［J］.Matrix Biol,2018,65:59-74.
［9］　WU H,CHEN W,ZHAO F,ZHOU Q,REINACH P S,DENG L,et al.Scleral hypoxia is a target for myopia control［J］.Proc Natl Acad Sci U S A,2018,115(30):e7091-e7100.
［10］　SHIELDS C L,SHIELDS J A.Recent developments in the management of retinoblastoma［J］.J Pediat Ophthalmol Strab,1999,36(1):8-36.
［11］　常鸣,贺司宇,岳娟,刘素素,王丽娅.mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响［J］.眼科新进展,2020,40(6):520-523.
CHANG M,HE S Y,YUE J,LIU S S,WANG L Y.Effects of rapamycin on corneal scarring in mice induced by fungal keratitis ［J］.Rec Adv Ophthalmol,2020,40(6):520-523.
［12］　张君娜,蔡静,宋华勇,吴涛.雷帕霉素联合肝动脉化疗栓塞术对兔肝移植瘤模型中相关因子表达的影响［J］.癌症进展,2020,18(9):897-900.
ZHANG J N,CAI J,SONG H Y,WU T.Effects of rapamycin combined with hepatic arterial chemoembolization on the expression of related factors in rabbit liver transplantation tumor model ［J］.Oncol Prog,2020,18(9):897-900.
［13］　李琳琳,郑祥雄.雷帕霉素对早期糖尿病大鼠肾脏TGF-β1、FN表达的影响［J］.中国老年学杂志,2014,34(19):5491-5493.
LI L L,ZHENG X X.Effects of rapamycin on the expression of TGF-β1 and FN in early diabetic rats ［J］.Chin J Gerontol,2014,34(19):5491-5493.
［14］　肖潇.雷帕霉素抑制肝纤维化病理进程的疗效及其机制［J］.肝脏,2019,24(10):1157-1160.
XIAO X.Effects of rapamycin on the pathological progression of liver fibrosis and its mechanism ［J］.Chin Hepatol,2019,24(10):1157-1160.
［15］　ZHAO F,ZHOU Q,REINACH P S,YaANG J,MA L,WANG X,et al.Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice［J］.Am J Pathol,2018,188(8):1754-1767.
［16］　张新,巨朝娟,张剑,赵燕.形觉剥夺性近视模型大鼠巩膜成纤维细胞中TGF-β1表达及Wnt/β-catenin信号通路的调控作用［J］.吉林大学学报（医学版）,2019,45(4):861-866.
ZHANG X,JU C J,ZHANG J,ZHAO Y.Expression of TGF-β1 in scleral fibroblasts and the regulation of Wnt/β-catenin signaling pathway in a rat model of shape-deprivation myopia［J］.J Jilin Univ (Med Ed),2019,45(4):861-866.
［17］　刘双珍,朱玉广,吴小影,李凤云,闵晓珊,许惠卓.TGF-β1在鸡形觉剥夺性近视眼中的表达及作用［J］.眼科研究,2005,23(1):55-57.
LIU S Z,ZHU Y G,WU X Y,LI F Y,MIN X S,XU H Z.Expression and role of TGF-β1 in chicken myopia ［J］.Chin Ophthalmic Res,2005,23(1):55-57.
［18］　LIU H,XIANG N,ZHANG H.Influence of high level TGF-beta1 on scleral thickness［J］.Huazhong Univ Sci Technol Med Sci,2007,27(5):601-604.
［19］　Van PUTTEN S,SHAFIEYAN Y,HINZ B.Mechanical control of cardiac myofibroblasts［J］.J Mol Cell Cardiol,2016,93:133-142.
［20］　GERARDUZZI C,DI BATTISTA J A.Myofibroblast repair mechanisms post-inflammatory response:a fibrotic perspective?［J］.Inflamm Res,2017,66(6):451-465.
［21］　WU P C,TSAI C L,GORDON G M,JEONG S,ITAKURA T,PATEL N,et al.Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice?［J］.Mol Vis,2015,21:138-147.
［22］　JOBLING A I,GENTLE A,METLAPALLY R,MCGOWAN B J,MCBRIEN N A.Regulation of scleral cell contraction by transforming growth factor-beta and stress:competing roles in myopic eye growth［J］.J Biol Chem,2009,284:2072-2079.
［23］　刘焰,胡道德,顾磊,许迅.Lewis鼠结膜下注射雷帕霉素的眼毒性研究［J］.眼科新进展,2008,28(7):498-500.
LIU Y,HU D D,GU L,XU X.Ophthalmic toxicity of subconjunctival injection of rapamycin in Lewis mice［J］.Rec Adv Ophthalmol,2008,28(7):498-500.
［24］　任静,秦波,邹畅,何靖,刘身文.雷帕霉素对缺氧损伤视网膜神经节细胞的保护作用及其机制［J］.中华实验眼科杂志,2017,35(5):404-409.
REN J,QIN B,ZOU C,HE J,LIU S W.Effects of rapamycin on retinal ganglion cells injured by hypoxia and its mechanism ［J］.Chin J Exp Ophthalmol,2017,35(5):404-409.
［25］　GIBBONS J J,ABRAHAM R T,YU K.Mammalian target of rapamycin:discovery of rapamycin reveals a signaling pathway important for normal and cancer cell growth［J］.Semin Oncol,2009,36（Suppl 3）:S3-S17.
［26］　CARAMS B,HASEGAWA A,TANIGUCHI N,MIYAKI S,BLANCO F J,LOTZ M.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis［J］.Ann Rheum Dis,2012,71(4):575-581.

相似文献/References:

[1]李勇子 刘桂香 王玲 隋爱玲 郭强强 万鲁芹.TIMP-2基因转染FDM豚鼠模型早期后极部巩膜中MMP-2的动态表达[J].眼科新进展,2012,32(12):000.
[2]李瑞凤 刘桂香 万鲁芹 颜晓萍 黄小凤 李勇子.TIMP-2基因转染 FDM 豚鼠后极部巩膜MMP-2蛋白早期动态表达[J].眼科新进展,2012,32(3):000.
[3]赵昆,黄敏丽,王淼,等.雷帕霉素对视网膜缺血-再灌注损伤的保护作用[J].眼科新进展,2014,34(5):409.[doi:10.13389/j.cnki.rao.2014.0113]
　ZHAO Kun,HUANG Min-Li,WANG Miao,et al.Protective effects of raamycin on retinal is-chemia-reperfusion injury of rats[J].Recent Advances in Ophthalmology,2014,34(3):409.[doi:10.13389/j.cnki.rao.2014.0113]
[4]魏红领,刘韶瑞,陆宁.雷帕霉素-壳聚糖-海藻酸钙缓释微球抑制青光眼滤过术区瘢痕增殖的实验研究[J].眼科新进展,2016,36(2):110.[doi:10.13389/j.cnki.rao.2016.0030]
　WEI Hong-Ling,LIU Shao-Rui,LU Ning.Experimental study on anti-proliferation of rapamycinchitosan-microspheres sustained release microspheres during glaucoma filtering surgery in rabbit[J].Recent Advances in Ophthalmology,2016,36(3):110.[doi:10.13389/j.cnki.rao.2016.0030]
[5]齐鹏,孙丹宇,包赫,等.雷帕霉素对高糖诱导人晶状体上皮细胞氧化应激的保护作用[J].眼科新进展,2018,38(3):235.[doi:10.13389/j.cnki.rao.2018.0054]
　QI Peng,SUN Dan-Yu,BAO He,et al.Rapamycin protects human lens epithelial cells against oxidative stress induced by high glucose[J].Recent Advances in Ophthalmology,2018,38(3):235.[doi:10.13389/j.cnki.rao.2018.0054]
[6]郭芳,魏瑞华,吴绵绵,等.近视动物模型及发病机制的研究进展[J].眼科新进展,2018,38(5):490.[doi:10.13389/j.cnki.rao.2018.0115]
　GUO Fang,WEI Rui-Hua,WU Mian-Mian,et al.Research advances in the animal models and pathogenic mechanisms of myopia[J].Recent Advances in Ophthalmology,2018,38(3):490.[doi:10.13389/j.cnki.rao.2018.0115]
[7]席亚慧,刘红玲,刘明月,等.mTOR-siRNA转染人晶状体上皮细胞对PI3K/AKT/mTOR信号通路蛋白p70S6K及4EBP1表达的影响[J].眼科新进展,2018,38(9):804.[doi:10.13389/j.cnki.rao.2018.0191]
　XI Ya-Hui,LIU Hong-Ling,LIU Ming-Yue,et al.The effect of mTOR-siRNA transfection of human lens epithelial cells on the expression of PI3K/AKT/mTOR signaling protein p70S6K and 4EBP1[J].Recent Advances in Ophthalmology,2018,38(3):804.[doi:10.13389/j.cnki.rao.2018.0191]
[8]展欣,朱子诚,孙思勤,等.激活蛋白1（AP-1）在豚鼠进展性近视巩膜重塑中与I型胶原的表达关系研究[J].眼科新进展,2019,39(2):129.[doi:10.13389/j.cnki.rao.2019.0029]
　ZHAN Xin,ZHU Zi-Cheng,SUN Si-Qin,et al.Expression changes of activator protein 1 and its correlation with collagen Ⅰ in the sclera remodeling of progressive myopia in guinea pigs[J].Recent Advances in Ophthalmology,2019,39(3):129.[doi:10.13389/j.cnki.rao.2019.0029]
[9]郭芳,魏瑞华,孙笑笑,等.Cochlin及其编码基因凝血因子C同源物（COCH）在形觉剥夺性近视豚鼠眼球后极部组织的表达[J].眼科新进展,2019,39(3):201.[doi:10.13389/j.cnki.rao.2019.0046]
　GUO Fang,WEI Rui-Hua,SUN Xiao-Xiao,et al.Expression of Cochlin and its coding gene coagulation factor C homology (COCH) in the ocular posterior polar tissues of guinea pigs with form-deprived myopia[J].Recent Advances in Ophthalmology,2019,39(3):201.[doi:10.13389/j.cnki.rao.2019.0046]
[10]常鸣,贺司宇,岳娟,等.mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响[J].眼科新进展,2020,40(6):520.[doi:10.13389/j.cnki.rao.2020.0119]
　CHANG Ming,HE Siyu,YUE Juan,et al.Effect of mTOR pathway inhibitor Rapamycin on corneal scarring in mice with fungal keratitis[J].Recent Advances in Ophthalmology,2020,40(3):520.[doi:10.13389/j.cnki.rao.2020.0119]

备注/Memo

备注/Memo:
山东省医药卫生科技发展计划项目（编号：2016WS0022）
更新日期/Last Update: 2021-03-05