昼夜节律变化对RORs表达及RORs激动剂SR1078对角膜上皮创伤修复的影响  

Effect of circadian rhythm changes on the expression of RORs and the RORs agonist SR1078 on corneal epithelial wound repair

作　　者：徐鹏洋 李志杰 薛芸霞 Xu Pengyang;Li Zhijie;Xue Yunxia(Department of Pathophysiology,Medical College of Jinan University,Guangzhou 510632,China;Institute of Ophthalmology,Medical College of Jinan University,Guangzhou 510632,China)

机构地区：[1]暨南大学基础医学院病理生理学系,广州510632 [2]暨南大学基础医学院眼科研究所,广州510632

出　　处：《中华实验眼科杂志》2022年第2期118-125,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81770962、81700808);广东省医学科研基金项目(A2020318)。

摘　　要：目的探讨昼夜节律变化对视黄酸相关孤儿受体(RORs)表达量及RORs激动剂SR1078对角膜上皮创伤修复的影响。方法选取SPF级6~8周龄C57BL/6雌性小鼠228只,采用随机数表法将其中180只小鼠分为昼夜节律正常组、全昼组、全夜组、昼夜颠倒12 h组和昼夜颠倒3周组,每组36只。将剩余48只小鼠采用随机数表法随机分为磷酸盐缓冲液(PBS)对照组和SR1078组,每组24只。按照分组,将小鼠置于可控制光照(光照强度300 lx)及黑暗时间的节律箱中,其中昼夜节律正常组、PBS对照组和SR1078组节律箱的光照时间为7:00~19:00,黑暗时间为19:00~次日7:00。根据Zeitgeber Time计时法,以开始光照时间7:00记为ZT0,以关闭光照时间19:00记为ZT12。采用实时荧光定量PCR法检测昼夜节律正常组、全夜组、全昼组、昼夜颠倒12 h组和昼夜颠倒3周组ZT1、ZT5、ZT9、ZT13、ZT17、ZT21各时间点RORα和RORγmRNA相对表达量。PBS对照组和SR1078组采用高尔夫样刀建立小鼠角膜上皮损伤模型并按照分组情况用相应试剂点眼,每隔6 h给药1次。采用Adobe Photoshop CC2019软件测量角膜上皮缺损面积,计算并比较2个组角膜上皮缺损率。分析昼夜节律正常组、全昼组、全夜组、昼夜颠倒12 h组和昼夜颠倒3周组小鼠角膜上皮RORα和RORγmRNA相对表达量与PBS对照组和SR1078组角膜上皮缺损率的相关性。采用全角膜铺片及免疫荧光染色法观察角膜上皮修复情况,计算并比较PBS对照组和SR1078组小鼠角膜上皮分裂细胞数量。结果与昼夜节律正常组比较,全昼组、全夜组、昼夜颠倒12 h组和昼夜颠倒3周组小鼠RORα/RORγmRNA相对表达量整体呈减少趋势。造模后不同时间点PBS对照组和SR1078组小鼠角膜上皮缺损率总体比较差异有统计学意义(F_(组别)=74.01,P<0.001;F_(时间)=5171.48,P<0.001),其中造模后12 h SR1078组角膜上皮缺损率显著低于PBS对照组,差异有统计学意义(P<0.05)。角膜�Objective To investigate the effect of circadian rhythm changes on the expression of retinoic acid-related orphan receptors(RORs)and the RORs agonist SR1078 on corneal epithelial wound repair.Methods A total of 228 SPF C57BL/6 female mice aged 6-8 weeks old were selected,and 180 mice were divided into the normal circadian rhythm group,full-day group,full-night group,12-hour reversed circadian rhythm group and 3-week reversed circadian rhythm group,with 36 mice in each group.The remaining 48 mice were randomly divided into phosphate buffered saline(PBS)control group and SR1078 group by random number table method,with 24 mice in each group.According to grouping,the mice were placed in a light box where the light(light intensity of 300 lx)and dark time could be controlled.The light time of the normal circadian rhythm group,the PBS control group and the SR1078 group in the light box was from 7:00 to 19:00,and the dark time was from 19:00 to 7:00 the next day.According to the Zeitgeber Time method,the starting time of light at 7:00 was recorded as ZT0,and the time of closing light at 19:00 was recorded as ZT12.Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of RORαand RORγmRNA at ZT1,ZT5,ZT9,ZT13,ZT17,ZT21 in the five groups.In the PBS control group and SR1078 group,a golf-like knife was used to establish the mouse corneal epithelial injury model,and the model eyes were administered with drugs once every 6 hours according to the grouping.The corneal epithelial defect area was measured with Adobe Photoshop CC2019 software,and the corneal epithelial defect rate was calculated and compared between the two groups.The correlation between the relative expression levels of RORαand RORγmRNA in mice corneal epithelium of the five groups and corneal epithelial defect rate in the PBS control group and SR1078 group was analyzed.The corneal epithelium repair was observed by whole cornea spreading and immunofluorescence staining,and the number of corneal epithelial

关 键 词：角膜 昼夜节律 创伤修复 SR1078 时钟基因 

分 类 号：R772.2[医药卫生—眼科]