CAJ | 학술논문

目的研究CREB1在氧诱导视网膜病变(OIR)小鼠模型视网膜新生血管形成中的表达变化,探寻视网膜新生血管形成的可能机制.方法实验研究.选用7日龄健康清洁级C57BL/6J小鼠134只,随机分为正常对照组(67只)和OIR模型组(67只).将小鼠与哺乳母鼠共同置于(75±2)％氧环境内饲养5d后转移至正常环境中饲养5d,建立小鼠OIR模型,17日龄的OIR鼠在空气中再继续饲养4d,正常对照组在正常氧环境中饲养21 d.出生后第17 d时取OIR模型组和正常对照组小鼠制备视网膜切片HE染色法和FITC-dextran心脏灌注视网膜铺片法观察视网膜新生血管情况,以及视网膜组织冰冻切片免疫荧光染色观察P-CREB1表达情况.取2组鼠第7、9、12、14、17、21 d的视网膜采用实时定量聚合酶链反应(real-time PCR)法和Western Blot法检测CREB1mRNA和蛋白在小鼠视网膜中的表达情况.数据分析采用独立样本t检验和两因素方差分析方法,两两比较采用Bonferronipost-test检验.结果 17 d时OIR模型组较正常对照组突破视网膜内界膜的内皮细胞核数明显增加(t=1 1.31,P＜0.05),且模型组视网膜新生血管区及无灌注区面积分别为(21.40±2.72)％和(30.61±3.12)％.与正常对照组相比,模型组小鼠视网膜中可见大量P-CREB1蛋白荧光,主要表达在内核层和神经节细胞层.Real-time PCR和Western Blot结果表明,正常对照组第7、9、12、14、17天的CREB1mRNA和蛋白在视网膜中的表达水平逐渐升高,21 d时开始出现下降的趋势;在各相应时间点OIR模型组里的CREB1相对表达量除第7天外,其余时间点均高于正常对照组,差异均有统计学意义(P＜0.05).结论 CREB1的表达水平与视网膜新生血管的形成存在时空对应关系,CREB1的高表达可能参与了OIR模型中视网膜新生血管的形成过程.
목적 연구CREB1재양유도시망막병변(OIR)소서모형시망막신생혈관형성중적표체변화,탐심시망막신생혈관형성적가능궤제.방법 실험연구.선용7일령건강청길급C57BL/6J소서134지,수궤분위정상대조조(67지)화OIR모형조(67지).장소서여포유모서공동치우(75±2)％양배경내사양5d후전이지정상배경중사양5d,건립소서OIR모형,17일령적OIR서재공기중재계속사양4d,정상대조조재정상양배경중사양21 d.출생후제17 d시취OIR모형조화정상대조조소서제비시망막절편HE염색법화FITC-dextran심장관주시망막포편법관찰시망막신생혈관정황,이급시망막조직빙동절편면역형광염색관찰P-CREB1표체정황.취2조서제7、9、12、14、17、21 d적시망막채용실시정량취합매련반응(real-time PCR)법화Western Blot법검측CREB1mRNA화단백재소서시망막중적표체정황.수거분석채용독립양본t검험화량인소방차분석방법,량량비교채용Bonferronipost-test검험.결과 17 d시OIR모형조교정상대조조돌파시망막내계막적내피세포핵수명현증가(t=1 1.31,P＜0.05),차모형조시망막신생혈관구급무관주구면적분별위(21.40±2.72)％화(30.61±3.12)％.여정상대조조상비,모형조소서시망막중가견대량P-CREB1단백형광,주요표체재내핵층화신경절세포층.Real-time PCR화Western Blot결과표명,정상대조조제7、9、12、14、17천적CREB1mRNA화단백재시망막중적표체수평축점승고,21 d시개시출현하강적추세;재각상응시간점OIR모형조리적CREB1상대표체량제제7천외,기여시간점균고우정상대조조,차이균유통계학의의(P＜0.05).결론 CREB1적표체수평여시망막신생혈관적형성존재시공대응관계,CREB1적고표체가능삼여료OIR모형중시망막신생혈관적형성과정.
Objective To study the relationship between the expression of CREB1 and retinal neovascularization (RNV) in a mouse model of oxygen-induced retinopathy (OIR).Methods Postnatal day 7 (P7) mice (n=134) were randomly assigned to two groups:a control group (n=67) and an OIR group (n=67).OIR was induced by exposing P7 mice to (75±2)％ O2 for 5 days,followed by exposure to room air for an additional 5 days.P17 OIR mice were raised in the normal environment for additional 4 days.The mice from the control group were raised in a normal environment for 21 days.The P17 mice from the two groups were sacrificed,and retinal sections for HE staining and flat mounts after cardiac perfusion with FITC-dextran were used to detect RNV.Immunofluorescence of the frozen retinal sections showed the expression and location of the P-CREB1 protein.Real-time PCR and Western Blot were used to detect the expression of CREB1 in the retina.Two-way ANOVA was used for data comparison of the two groups at different time points and a Bonferroni post-test was used for comparison between two time points within a group.An independent samples t test at the same time points between the groups was used for statistical analysis.Results The number of cellular nuclei in the vascular endothelium breaking through the retinal internal limiting membrane was significantly higher in the OIR group than in the control group at P17 (t=11.31,P＜0.05).Areas of new retinal blood vessels and avascular zones were (21.40±2.72)％ and (30.61±3.12)％,respectively,in OIR mice at P17 P-CREB1 protein was expressed more strongly in the inner nuclear layer and ganglion cell layer of the retinas in the OIR group than in the control group.The results of real-time PCR and Western Blot showed that the relative expression levels of mRNA and CREB1 protein gradually increased from P7 to P17,followed by decreased expression at P21 in the control group.But the values were significantly higher in the OIR group than in the control group in mice of all ages except for P7 mice (P＜0.05).Conclusion These results indicate a space-time corresponding relationship between the expression of CREB1 and RNV.The overexpression of CREB1 could be involved in the process of RNV in the OIR model.