多聚嘧啶序列结合蛋白相关剪接因子对缺氧诱导人视网膜微血管内皮细胞功能的影响  

Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxiainduced human retinal microvascular endothelial cells

作　　者：徐嫚鸿 王林妮 林婷婷 任新军 柯屹峰 胡立颖 焦明菲 王勇[1] 王琼 洪雅茹 李筱荣[1] 东莉洁[1] Xu Manhong;Wang Linni;Lin Tingting;Ren Xinjun;Ke Yifeng;Hu Liying;Jiao Mingfei;Wang Yong;Wang Qiong;Hong Yaru;Li Xiaorong;Dong Lijie(Tianjin Medical University Eye Hospital,Tianjin Medical University Eye Institute,College of Optometry and Ophthalmology,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院,天津医科大学眼科研究所,天津医科大学眼视光学院,300384

出　　处：《中华眼底病杂志》2020年第2期135-142,共8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(2017KJ216、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)。

摘　　要：目的观察多聚嘧啶序列结合蛋白相关剪接因子(PSF)对缺氧诱导的人视网膜微血管内皮细胞(hRMECs)功能的影响。方法采用三质粒系统构建慢病毒颗粒(LV)-PSF。LV-PSF体外感染hRMECs后通过流式细胞计数法测定其感染效率。应用实时定量PCR(RT-PCR)检测经LV-PSF感染的hRMECs中PSF mRNA表达水平。将实验分为体内和体外两部分。体内实验:7日龄健康C57B/L6小鼠20只,采用随机数字表法分为正常组、氧诱导视网膜病变(OIR)组、OIR+LV-空载体(Vec)组和OIR+LV-PSF组,每组5只。除正常组外,其余3组构建OIR模型。OIR组除缺氧刺激外,不做其余处理。OIR+LV-Vec组和OIR+LV-PSF组小鼠分别玻璃体腔注射LV-Vec或LV-PSF。观察LV-PSF对视网膜新生血管(RNV)形成的影响。体外实验:将hRMECs分为正常组、缺氧组、空载组、PSF高表达组。正常组为正常体外培养的hRMECs;缺氧组为缺氧刺激3 h恢复正常培养条件24 h的hRMECs;空载组、PSF高表达组分别为用LV-Vec、LV-PSF感染48 h,再用缺氧刺激3 h恢复正常培养条件24 h的hRMECs。采用MTT比色法观察PSF对细胞增生能力的影响。采用细胞划痕实验和Transwell迁移实验观察PSF对缺氧刺激下细胞迁移能力的影响。采用RT-PCR观察各组细胞中HIF-1α、VEGF及PSF的mRNA表达。结果成功构建可稳定高表达PSF的LV-PSF,流式细胞仪测定其感染效率为97%,RT-PCR测得经LV-PSF感染的hRMECs中PSF mRNA水平明显上调。体内实验:OIR组、OIR+LV-Vec组小鼠RNV面积较正常组明显增加(t=18.31、43.71),OIR+LV-PSF组小鼠RNV面积较OIR组(t=11.30)、OIR+LV-Vec组(t=15.47)明显减小,差异均有统计学意义(P<0.05)。体外实验:MTT比色法检测结果显示,缺氧组hRMECs增生能力较正常组明显增强(t=2.57),PSF高表达组hRMECs增生能力较正常组、缺氧组、空载组明显降低(t=5.26、5.46、3.73),差异均有统计学意义(P<0.05)。细胞划痕实验结果显示,缺氧刺激3 h恢复正常条件24 h或48Objective To observe the effect of pyrimidine bundle-binding protein-associated splicing factors(PSF)on the function of hypoxia-induced human retinal microvascular endothelial cells(hRMECs).Methods A three-plasmid system was used to construct lentivirus(LV)-PSF.After LV-PSF infected hRMECs in vitro,the infection efficiency was measured by flow cytometry.Real-time quantitative PCR(RT-PCR)was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF.The experiment was divided into two parts,in vivo and in vitro.In vivo experiments:20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group,oxygen-induced retinopathy(OIR)group,OIR+LV-Vec group,and OIR+LPSF group,each group has five mice.Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated.The mice in the OIR+LV-Vec group and the OIR+LV-PSF group were injected with an empty vector(LV-Vec)or LV-PSF in the vitreous cavity,respectively.The effect of LV-PSF on the formation of retinal neovascularization(RNV)was observed then.In vitro experiments:hRMECs were divided into normal group,hypoxia group,vector group,and PSF high expression group.HRMECs in the normal group were cultured in vitro;hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation;hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h,and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h.The effect of PSF on cell proliferation was observed by MTT colorimetry.Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation.RT-PCR was used to observe the mRNA expression of HIF-lα,VEGF and PSF in each group of cells.Results The LV-PSF of stably expressing PSF was successfully constructed.The infection efficiency was 97%determined by flow cytometry.The level of PSF mRNA in hRMECs infe

关 键 词：多聚嘧啶区结合蛋白质 视网膜新生血管化 缺氧 视网膜血管/细胞学 内皮细胞/生理学 细胞 培养的 

分 类 号：R774[医药卫生—眼科]