摘要
目的观察慢病毒介导聚嘧啶束结合蛋白相关剪接因子(PSF)对氧诱导视网膜病变(OIR)小鼠视网膜新生血管的抑制作用。方法5日龄C57BL/6J小鼠112只随机分为正常对照组、单纯OIR模型组、OIR模型+慢病毒空载体处理组(以下简称Vec组)及OIR模型+PSF慢病毒处理组(以下简称PSF组),分别为16、32、32、32只。小鼠7日龄时,正常对照组小鼠常规环境饲养;单纯OIR模型组、Vec组及PSF组小鼠建立OIR模型。小鼠12日龄时,Vec组、PSF组小鼠玻璃体腔分别注射滴度为1×1011 TU/ml的空载体病毒或PSF慢病毒1μl。正常对照组和单纯OIR模型组小鼠不再做任何处理。小鼠17日龄时,采用HE染色计数突破视网膜内界膜的血管内皮细胞核;作视网膜铺片,测量各组小鼠视网膜无灌注区相对面积;采用实时定量PCR检测各组小鼠视网膜中NF-E2相关因子2(Nrf2)和血红素氧合酶1(HO-1)的mRNA相对表达量;采用Western blot检测各组小鼠视网膜中Nrf2、HO-1及PSF的蛋白相对表达量。组间比较采用单因素方差分析。结果正常对照组、单纯OIR模型组、Vec组、PSF组小鼠突破内界膜的血管内皮细胞核数分别为0.00、14.36±5.50、15.67±4.96、8.13±2.09个;视网膜无灌注区面积分别为0.00%、(35.71±2.81)%、(36.57±4.53)%、(15.33±4.75)%。4组间小鼠突破内界膜的血管内皮细胞核数及视网膜无灌注区面积比较,差异均有统计学意义(F=24.87、165.70,P<0.05)。组间两两比较,单纯OIR模型组、Vec组较正常对照组突破内界膜的血管内皮细胞核数增多,视网膜无灌注区面积增大;PSF组较单纯OIR模型组、Vec组突破内界膜的血管内皮细胞核数减少,视网膜无灌注区面积减小,差异均有统计学意义(P<0.05)。实时定量PCR及Western blot检测结果显示,4组间小鼠视网膜Nrf2、PSF mRNA相对表达量(F=53.66、83.54)以及Nrf2、HO-1、PSF蛋白相对表达量(F=58.38、52.69、24.79)比较,差异均有统计学意义(P<0.05)。组间两两比较,单纯OIR模型组、Vec组小鼠视网膜Nrf2、PSF mRNA相对表达量及Nrf2、HO-1、PSF蛋白相对表达量较正常对照组明显降低,PSF组小鼠视网膜Nrf2、PSF mRNA相对表达量及Nrf2、HO-1、PSF蛋白相对表达量较单纯OIR模型组、Vec组明显增加,差异均有统计学意义(P<0.05)。结论慢病毒介导的PSF可通过上调Nrf2及HO-1的表达抑制OIR小鼠视网膜新生血管形成。
Objective To investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor(PSF)on retinal neovascularization(RNV)in mice model of oxygeninduced retinopathy(OIR).Methods One hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group,simple OIR model group,OIR model+lentivirus empty vector treatment group(Vec group)and OIR model+PSF lentivirus treatment group(PSF group),with 16,32,32 and 32 mice,respectively.When the mice were 7 days old,the mice in the normal control group were fed in a routine environment,and the mice in the OIR model group,Vec group and PSF group were established OIR model.The mice in the Vec group and PSF group were given an intravitreal injection of 1μl of lentiviral vector and PSF lentivirus(titer 1×1011 TU/ml)at the age of 12 days.No injection was performed in the normal control group and simple OIR group.RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina.Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2(Nrf2)and hemeoxygenase-1(HO-1).Western blot analysis was applied to detect the protein expression of Nrf2,HO-1 and PSF.Results Of the normal control group,simple OIR model group,Vec group and PSF group,the number of pre-retinal neovascular cell nuclei were 0.00,14.36±5.50,15.67±4.96,8.13±2.09,the non-perfusion area were 0.00%,(35.71±2.81)%,(36.57±4.53)%,(15.33±4.75)%,respectively.The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant(F=24.87,165.70;P<0.05).Compared with the normal control group,there were more pre-retinal neovascular cell nucleis and larger nonperfusion area in the simple OIR model group and Vec group(P<0.05).Compared with the simple OIR model group and Vec group,there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group(P<0.05).Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2,HO-1(F=53.66,83.54)and protein expression of Nrf2,HO-1 and PSF(F=58.38,52.69,24.79)among 4 groups were significant(P<0.05).The mRNA expression of Nrf2,HO-1 and protein expression of Nrf2,HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group(P<0.05).The mRNA expression of Nrf2,HO-1 and protein expression of Nrf2,HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group(P<0.05).model group and Vec group(P<0.05).Conclusion Intravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
作者
黄亮瑜
柯屹峰
林婷婷
步绍翀
任新军
焦明菲
王勇
胡立颖
王琼
洪雅茹
李筱荣
东莉洁
Huang Liangyu;Ke Yifeng;Lin Tingting;Bu Shaochong;Ren Xinjun;Jiao Mingfei;Wang Yong;HuLiying;Wang Qiong;Hong Yaru;Li Xiaorong;Dong Lijie(Tianjin Medical University Eye Hospital,Tianjin Medical University Eye Institute,The College of Optometry&Ophthalmology,Tianjin 300384,China;Department of Ophthalmology,The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300381,China)
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2020年第1期53-59,共7页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金(81570872)
天津市卫计委青年医学新锐人才项目
天津医科大学眼科医院青年创新人才项目(YDYYRCXM-C2018-01/02/03)
天津市教委科研计划一般项目(2017KJ214、2018KJ051)
天津市临床重点学科(专科)天津医科大学眼科医院青年项目(TJLCZDXKQ024、TJLCZDXKQ017)
白求恩朗沐中青年眼科科研基金(BJ-LM2018005J)。
