中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2009年
3期
199-205
,共7页
闵晓洁%周庆军%刘廷%银红梅%董晓光%谢立信
閔曉潔%週慶軍%劉廷%銀紅梅%董曉光%謝立信
민효길%주경군%류정%은홍매%동효광%사립신
高氧症%视网膜新生血管化%端粒,未端转移酶%内皮细胞%基因表达调控
高氧癥%視網膜新生血管化%耑粒,未耑轉移酶%內皮細胞%基因錶達調控
고양증%시망막신생혈관화%단립,미단전이매%내피세포%기인표체조공
Hyperoxia%Retinal neovascularization%Telomerase%Endothelial cells%Gene expression regulation
目的 研究高氧诱导视网膜新生血管模型中小鼠端粒酶逆转录酶(TERT)基因表达水平是否有变化,为进一步研究视网膜新生血管疾病的预防和治疗提供新的靶点.方法 实验研究.选取7 d龄C57BL/6J新生小鼠32只,分高氧组和对照组,每组16只.高氧组小鼠以密闭氧箱内以75%±2%氧浓度饲养5 d后置于正常氧浓度环境中,正常对照组小鼠于正常氧环境中饲养.于小鼠生后12、14及19 d时分别取高氧组和对照组小鼠各2只(4只眼),经尾静脉行2%伊文思蓝溶液灌注并做视网膜铺片,荧光显微镜下观察视网膜新生血管的形成情况.高氧模型组和正常对照组生后19 d幼鼠3只,行HE染色,光学显微镜下观察视网膜血管形态,观察突破内界膜的内皮细胞核数.取生后19 d高氧组和对照组小鼠,分别取其视网膜组织并提取总RNA,反转录成cDNA后行反转录PCR,2%琼脂糖凝胶电泳并照相.提取视网膜总RNA,反转录成cDNA后(同RT-PCR),配制荧光定量实时PCR反应体系(总计20μl),在60℃检测荧光信号,分析图像.分别取高氧模型组和正常对照组P19小鼠行眼球切片,常规处理后TERT抗体孵育37℃ 60 min,HRP酶标二抗孵育30 min,DAB显色,中性胶封片,镜下观察并照相.结果高氧诱导模型小鼠牛后12 d眼底后极部出现大片无灌注区,生后14 d眼底后极部出现新牛血管迂曲、渗漏等视网膜血管病变.生后17～19 d视网膜新生血管形成达到高峰.正常小鼠视网膜组织切片HE染色基本看不剑突出内界膜的血管芽及血管管腔,内界膜下视网膜内的血管内皮细胞核散在分布、数量较少;高氧组见大量突出内界膜伸向玻璃体腔的血管管腔及血管芽,内界膜下视网膜内也有大量血管内皮细胞增生.19 d高氧模型组小鼠视网膜TERT及bFGF mRNA表达较同日龄正常对照组小鼠明显提高,二者差异有统计学意义(F=8.575,5.667;P＜0.05).生后19 d实时PCR检测高氧模型组小鼠视网膜TERT mRNA表达较同日龄正常对照组小鼠明显上调,差异有统计学意义(F=173.104,P＜0.05).生后19 d高氧诱导小鼠视网膜新生血管模型中视网膜新生血管TERT表达阳性,同日龄对照组新生小鼠视网膜血管TERT表达阴性.结论高氧诱导视网膜新生血管小鼠模型中端粒酶逆转录酶和新生血管形成相关因子表达水平明显上调,可能会成为视网膜新生血管疾病预防和治疗的新靶点.(中华眼科杂志,2009,45:199-205)
目的 研究高氧誘導視網膜新生血管模型中小鼠耑粒酶逆轉錄酶(TERT)基因錶達水平是否有變化,為進一步研究視網膜新生血管疾病的預防和治療提供新的靶點.方法 實驗研究.選取7 d齡C57BL/6J新生小鼠32隻,分高氧組和對照組,每組16隻.高氧組小鼠以密閉氧箱內以75%±2%氧濃度飼養5 d後置于正常氧濃度環境中,正常對照組小鼠于正常氧環境中飼養.于小鼠生後12、14及19 d時分彆取高氧組和對照組小鼠各2隻(4隻眼),經尾靜脈行2%伊文思藍溶液灌註併做視網膜鋪片,熒光顯微鏡下觀察視網膜新生血管的形成情況.高氧模型組和正常對照組生後19 d幼鼠3隻,行HE染色,光學顯微鏡下觀察視網膜血管形態,觀察突破內界膜的內皮細胞覈數.取生後19 d高氧組和對照組小鼠,分彆取其視網膜組織併提取總RNA,反轉錄成cDNA後行反轉錄PCR,2%瓊脂糖凝膠電泳併照相.提取視網膜總RNA,反轉錄成cDNA後(同RT-PCR),配製熒光定量實時PCR反應體繫(總計20μl),在60℃檢測熒光信號,分析圖像.分彆取高氧模型組和正常對照組P19小鼠行眼毬切片,常規處理後TERT抗體孵育37℃ 60 min,HRP酶標二抗孵育30 min,DAB顯色,中性膠封片,鏡下觀察併照相.結果高氧誘導模型小鼠牛後12 d眼底後極部齣現大片無灌註區,生後14 d眼底後極部齣現新牛血管迂麯、滲漏等視網膜血管病變.生後17～19 d視網膜新生血管形成達到高峰.正常小鼠視網膜組織切片HE染色基本看不劍突齣內界膜的血管芽及血管管腔,內界膜下視網膜內的血管內皮細胞覈散在分佈、數量較少;高氧組見大量突齣內界膜伸嚮玻璃體腔的血管管腔及血管芽,內界膜下視網膜內也有大量血管內皮細胞增生.19 d高氧模型組小鼠視網膜TERT及bFGF mRNA錶達較同日齡正常對照組小鼠明顯提高,二者差異有統計學意義(F=8.575,5.667;P＜0.05).生後19 d實時PCR檢測高氧模型組小鼠視網膜TERT mRNA錶達較同日齡正常對照組小鼠明顯上調,差異有統計學意義(F=173.104,P＜0.05).生後19 d高氧誘導小鼠視網膜新生血管模型中視網膜新生血管TERT錶達暘性,同日齡對照組新生小鼠視網膜血管TERT錶達陰性.結論高氧誘導視網膜新生血管小鼠模型中耑粒酶逆轉錄酶和新生血管形成相關因子錶達水平明顯上調,可能會成為視網膜新生血管疾病預防和治療的新靶點.(中華眼科雜誌,2009,45:199-205)
목적 연구고양유도시망막신생혈관모형중소서단립매역전록매(TERT)기인표체수평시부유변화,위진일보연구시망막신생혈관질병적예방화치료제공신적파점.방법 실험연구.선취7 d령C57BL/6J신생소서32지,분고양조화대조조,매조16지.고양조소서이밀폐양상내이75%±2%양농도사양5 d후치우정상양농도배경중,정상대조조소서우정상양배경중사양.우소서생후12、14급19 d시분별취고양조화대조조소서각2지(4지안),경미정맥행2%이문사람용액관주병주시망막포편,형광현미경하관찰시망막신생혈관적형성정황.고양모형조화정상대조조생후19 d유서3지,행HE염색,광학현미경하관찰시망막혈관형태,관찰돌파내계막적내피세포핵수.취생후19 d고양조화대조조소서,분별취기시망막조직병제취총RNA,반전록성cDNA후행반전록PCR,2%경지당응효전영병조상.제취시망막총RNA,반전록성cDNA후(동RT-PCR),배제형광정량실시PCR반응체계(총계20μl),재60℃검측형광신호,분석도상.분별취고양모형조화정상대조조P19소서행안구절편,상규처리후TERT항체부육37℃ 60 min,HRP매표이항부육30 min,DAB현색,중성효봉편,경하관찰병조상.결과고양유도모형소서우후12 d안저후겁부출현대편무관주구,생후14 d안저후겁부출현신우혈관우곡、삼루등시망막혈관병변.생후17～19 d시망막신생혈관형성체도고봉.정상소서시망막조직절편HE염색기본간불검돌출내계막적혈관아급혈관관강,내계막하시망막내적혈관내피세포핵산재분포、수량교소;고양조견대량돌출내계막신향파리체강적혈관관강급혈관아,내계막하시망막내야유대량혈관내피세포증생.19 d고양모형조소서시망막TERT급bFGF mRNA표체교동일령정상대조조소서명현제고,이자차이유통계학의의(F=8.575,5.667;P＜0.05).생후19 d실시PCR검측고양모형조소서시망막TERT mRNA표체교동일령정상대조조소서명현상조,차이유통계학의의(F=173.104,P＜0.05).생후19 d고양유도소서시망막신생혈관모형중시망막신생혈관TERT표체양성,동일령대조조신생소서시망막혈관TERT표체음성.결론고양유도시망막신생혈관소서모형중단립매역전록매화신생혈관형성상관인자표체수평명현상조,가능회성위시망막신생혈관질병예방화치료적신파점.(중화안과잡지,2009,45:199-205)
Objective To establish oxygen-induced retinal neovascularization in mice and to detect the expression of mTERT in mice.Methods It was an experimental study Establishment of oxygen-induced retinal neovascularization in mice.Thirty-two 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group without restriction of gender.In oxygen-induced retinopathy group,16 mice were exposed to 75%±2% oxygen for 5 days and then to room air;In control group,16 mice were raised in room air.Observation of the retinal neovascularization.On the postnatal day 19,The mice's vena caudalis were perfused with 2% Evens blue solution.Eyeballs were enucleated and fixed in 4% paraformaldehyde for half an hour.Then the retina was separated and flat-mounted on the slide.The morphologic changes of retinal vessel were observed and captured under fluorescence microscope.Histological observation and vascular endothelial cells counting.The eyeballs were enucleated and then fixed.After paraffin imbedding,4μm serial slices,hematoxylin-eosin staining,select one section every 60 μm to count the endothelial cell nucleus that break through the inner limiting membrane.Expression of mTERT mRNA were confirmed by reverse-transcription polymerase chain reaction(RT-PCR).In the each group,the retina were all carefully dissected on the postnatal day 19.The total RNA wag isolated and cDNA was synthesized before RT-PCR was performed.The PCR products were separated by 2% agarose gel electrophoresis and photographed.Expression of mTERT mRNA were confirmed by Real-time PcR The total RNA was isolated and cDNA was synthesized(The same procedure as RT-PCR).Fiuorescent real-time quantitative polymerase chain reaction system(total 20μl)was made.The Fluorescent signals were detected at 60℃.The expression of mTERT were confirmed by immunohistochemistry.At P19,4μm cross sections were made in the hyperoxia-exposed and normal retinas.Sections were incubated with rabbit anti Human/Mouse/Rat Telomerase 60 minutes at 37℃.Anti-rabbit immunoglobulin G,depending on the primary antibody.was used as a secondary antibody for 30 min.Peroxidase activity was detected with the substrate diaminobenzidine.Permanent slides were covered with a 1.5 mm thick cover slip.examined using a light microscope and photographed.Results The centrel retina was nonperfused region at P12.The most of the central retina showed almost no perfusion and the radial vessels appeared tortuous and dilated at P14.Retinal neovascularization occurred at maximum between postnatal day 17 and postnatal day 19.Paraffin tissue slice with hematoxylin-eosin staining showed that in the control group the average counts of vascular endothelial cells which break through the inner limiting membrane were hardly seen,but in hyperxia group were noticeably more than in the control group.Reverse-transcription polymerase chain reaction(RT-PCR)resuls:the mRNA of mTERT and bFGF in the retinopathy group were higber than in the control group(P＜0.05).Real-time PCR results:the expression of mTERT mRNA in the retinopathy group was noticeably higher than in the control group(F=173.104,P＜0.05).Immunohistochemical staining showed that mTERT protein were positive in the retinal neovascularization of the hyperxia group,but were negative in the retinal vessel of the control group.Conclusions Telomerase reverse transcription and angiogenic correlation factors were up-regulated in a mouse model of oxygen-induced retinopathy,which may have therapeutic potential in the treatment with the neovsscularization in retinopathy.(Chin J Ophthalmol,2009,45:199-205)
