摘要
目的 分析氧诱导视网膜病变(OIR)小鼠模型的微小RNA表达谱,筛选可能与视网膜新生血管(RNV)形成有关的微小RNA.方法 7日龄健康C57BL/6J小鼠52只随机分为正常对照组和OIR组,每组26只.OIR组建立OIR小鼠模型;正常对照组不做任何处理.小鼠17日龄时,行视网膜铺片,观察视网膜血管形态;行视网膜石蜡切片,计数突破内界膜的视网膜血管内皮细胞核数;行微小RNA芯片分析,检测具有差异表达的微小RNA,再应用实时聚合酶链反应(RT-PCR)进行验证.结果 正常对照组小鼠视网膜血管平滑、均匀有序分布;OIR组小鼠周边视网膜血管纡曲、紊乱并伴有新生血管芽,中央视网膜无灌注区明显.正常对照组、OIR组小鼠视网膜无灌注区的相对面积分别为(6.57±3.6)%、(25.81±2.12)%;OIR组小鼠视网膜无灌注区的相对面积较正常对照组明显增大,差异有统计学意义(t=28.71,P<0.001).光学显微镜观察发现,正常对照组小鼠内界膜结构完整、平滑,血管内皮细胞排列整齐,偶见突破内界膜的血管内皮细胞核.正常对照组、OIR组平均每张切片突破内界膜的视网膜血管内皮细胞核数分别为(0.16±0.31)、(28.41±4.01)个.两组平均每张切片突破内界膜的视网膜血管内皮细胞核数比较,差异有统计学意义(t=54.45,P<0.001).微小RNA芯片分析结果显示,与正常对照组比较,OIR组有21个微小RNA的表达发生了1.5倍以上的变化.其中,上调者9个,下调者12个.差异表达倍数≥3.0的微小RNA为miR-3078、miR-140、miR-29b、miR-29c.RT-PCR检测发现,差异表达倍数≥3.0的4个微小RNA,其表达趋势与芯片分析结果一致.与正常对照组比较,OIR组miR-3078的表达明显上调,差异有统计学意义(t=-2.380,P<0.05);miR-140、miR-29b、miR-29c的表达明显下调,差异也有统计学意义(t=2.638、2.323、2,415,P<0.05).结论 OIR小鼠模型的微小RNA表达谱中,miR-3078、miR-140、miR-29b、miR-29c的差异表达倍数≥3.0,其可能与RNV形成有关.
Objective To study morphological characteristics and microRNA (miR) expression profiling in a mouse model of oxygen-induced retinopathy (OIR).Methods Healthy C57BL/6J female mice and pups were randomly divided into normal and O1R group at postnatal day 7 (P7).The normal group was raised in a conventional cage and exposed to room air for 10 days.The OIR group was raised in a sealed chamber and exposed to (75±2)% oxygen.The moms were alternated between the two groups every day to promote their survival under hyperoxia.The OIR group was returned to the room air at P12.At P17,mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran),the eye balls were fixed in 4% paraformaldehyde,and the retinal whole mounts were prepared.The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope; the eye balls were also processed for paraffin sections and Hematoxylin and Eosin (H&E) staining.The cell nucleus in the newly-formed vessels beyond the inner limiting membrane was quantified.The miR was extracted from the eyes,reverse transcribed,and subjected to a customized miR array analysis.The realtime PCR was preformed to verify the results of the miR array.Results Retinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous,disorganized with neovascular buds,and the avascular area was prominent in central retina.In contrast,the vessels were smooth,organized,and evenly distributed in the retinas of normal group.The percentage of avascular area in total retina area in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P< 0.01,normal group vs OIR group).H&E staining showed that the number of the cell nuclei beyond inner limiting membrane was (28.41 ±4.01) in OIR retina,which was substantially higher than that (0.16± 0.31) in normal retina (P<0.01,normal group vs OIR group).More interestingly,the results of miR array showed that 21 out of the 80 miRs examined exhibited more than 1.5-fold changes at expression level.Among these 21 miRs,9 were up-regulated,12 were down-regulated; 4 miRs showed more than 3-fold expression changes,3 were down-regulated and 1 was up-regulated.The expression of the 4 miRs was verified by real-time PCR.The expression trends of miR-3078,miR-140,miR-29b and miR-29c were consistent with those revealed by the miR array.MiR-3078 was significantly up-regulated (t=-2.380,P< 0.05.normal group vs OIR group),and the other 3 miRs were significantly down-regulated (t=2.638,2.323,2.415,P<0.05.normal group vs OIR group).Conclusions The OIR mouse model has been established in our study.Differential expression of the microRNAs,including miR-3078,140,29b and 29c,was detected in normal and OIR mouse retinas.These miR expression changes may be associated with retinal neovascularization.These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2015年第1期77-82,共6页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金(31100991)
留学回国人员科研启动基金
教育部博士点基金(20121202120005)