CAJ | 학술논문

目的观察色素上皮衍生因子(PEDF)对氧诱导视网膜新生血管(OIR)的抑制作用,初步探讨PEDF抑制新生血管形成的可能机制.方法 7日龄C57BL/6J新生小鼠140只,随机数字表法分为正常对照组、OIR模型组、PEDF治疗组、磷酸盐缓冲液(PBS)干预对照组.除正常对照组外,其余各组小鼠与哺乳母鼠共同置于氧浓度为(75±2)％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型;正常对照组小鼠始终置于正常氧环境饲养;OIR模型组小鼠出氧箱后不作任何处理.12、14日龄时,PEDF治疗组小鼠玻璃体注射分别注射2 μg/A的PEDF 1 μl;PBS干预对照组小鼠玻璃体腔注射等量PBS.视网膜铺片、冰冻切片荧光染色观察视网膜新生血管生长情况;蛋白免疫印迹法(Western blot)观察小鼠视网膜白细胞介素(IL)-1β蛋白表达量;实时逆转录-聚合酶链反应(RT-PCT)检测小鼠视网膜IL-1β mRNA相对表达量.结果视网膜铺片检查结果显示,OIR模型组视网膜新生血管面积较正常对照组明显增大,差异有统计学意义(t=15.02,P＜0.01);PEDF治疗组视网膜新生血管面积较PBS干预对照组明显减小,差异有统计学意义(t=5.96,P＜0.01).荧光显微镜观察结果显示,OIR模型组视网膜可见外丛状层(OPL)与神经节细胞层(GCL)之间的新生血管簇较正常对照组明显增多;PEDF治疗组视网膜OPL与GCL之间的新生血管簇较PBS干预对照组明显减少.Western blot检测结果显示,OIR模型组视网膜IL-1β蛋白表达水平较正常对照组显著提高,差异均有统计学意义(t=3.35,P＜0.05).PEDF治疗组与PBS干预对照组比较,IL-1β蛋白表达水平下降,差异有统计学意义(P＜0.05).正常对照组与PEDF治疗组比较,IL-1β蛋白表达水平无显著差异(F=11.764,P＞0.05).实时RT-PCR检测结果显示,OIR模型组视网膜IL-1βmRNA相对表达量较正常对照组明显增加,差异有统计学意义(t=4.43,P＜0.01).PEDF治疗组与PBS干预对照组比较,前者IL-1β mRNA相对表达量显著减少,差异有统计学意义;正常对照组与PEDF治疗组比较,IL-1β mRNA相对表达量无显著差异(F=11.151,P＞0.05).结论 PEDF可抑制OIR视网膜新生血管的形成.下调IL-1β在视网膜的表达可能是其机制之一. 目的觀察色素上皮衍生因子(PEDF)對氧誘導視網膜新生血管(OIR)的抑製作用,初步探討PEDF抑製新生血管形成的可能機製.方法 7日齡C57BL/6J新生小鼠140隻,隨機數字錶法分為正常對照組、OIR模型組、PEDF治療組、燐痠鹽緩遲液(PBS)榦預對照組.除正常對照組外,其餘各組小鼠與哺乳母鼠共同置于氧濃度為(75±2)％的氧箱內飼養5d後轉移至正常環境中飼養5d,建立OIR模型;正常對照組小鼠始終置于正常氧環境飼養;OIR模型組小鼠齣氧箱後不作任何處理.12、14日齡時,PEDF治療組小鼠玻璃體註射分彆註射2 μg/A的PEDF 1 μl;PBS榦預對照組小鼠玻璃體腔註射等量PBS.視網膜鋪片、冰凍切片熒光染色觀察視網膜新生血管生長情況;蛋白免疫印跡法(Western blot)觀察小鼠視網膜白細胞介素(IL)-1β蛋白錶達量;實時逆轉錄-聚閤酶鏈反應(RT-PCT)檢測小鼠視網膜IL-1β mRNA相對錶達量.結果視網膜鋪片檢查結果顯示,OIR模型組視網膜新生血管麵積較正常對照組明顯增大,差異有統計學意義(t=15.02,P＜0.01);PEDF治療組視網膜新生血管麵積較PBS榦預對照組明顯減小,差異有統計學意義(t=5.96,P＜0.01).熒光顯微鏡觀察結果顯示,OIR模型組視網膜可見外叢狀層(OPL)與神經節細胞層(GCL)之間的新生血管簇較正常對照組明顯增多;PEDF治療組視網膜OPL與GCL之間的新生血管簇較PBS榦預對照組明顯減少.Western blot檢測結果顯示,OIR模型組視網膜IL-1β蛋白錶達水平較正常對照組顯著提高,差異均有統計學意義(t=3.35,P＜0.05).PEDF治療組與PBS榦預對照組比較,IL-1β蛋白錶達水平下降,差異有統計學意義(P＜0.05).正常對照組與PEDF治療組比較,IL-1β蛋白錶達水平無顯著差異(F=11.764,P＞0.05).實時RT-PCR檢測結果顯示,OIR模型組視網膜IL-1βmRNA相對錶達量較正常對照組明顯增加,差異有統計學意義(t=4.43,P＜0.01).PEDF治療組與PBS榦預對照組比較,前者IL-1β mRNA相對錶達量顯著減少,差異有統計學意義;正常對照組與PEDF治療組比較,IL-1β mRNA相對錶達量無顯著差異(F=11.151,P＞0.05).結論 PEDF可抑製OIR視網膜新生血管的形成.下調IL-1β在視網膜的錶達可能是其機製之一.
목적 관찰색소상피연생인자(PEDF)대양유도시망막신생혈관(OIR)적억제작용,초보탐토PEDF억제신생혈관형성적가능궤제.방법 7일령C57BL/6J신생소서140지,수궤수자표법분위정상대조조、OIR모형조、PEDF치료조、린산염완충액(PBS)간예대조조.제정상대조조외,기여각조소서여포유모서공동치우양농도위(75±2)％적양상내사양5d후전이지정상배경중사양5d,건립OIR모형;정상대조조소서시종치우정상양배경사양;OIR모형조소서출양상후불작임하처리.12、14일령시,PEDF치료조소서파리체주사분별주사2 μg/A적PEDF 1 μl;PBS간예대조조소서파리체강주사등량PBS.시망막포편、빙동절편형광염색관찰시망막신생혈관생장정황;단백면역인적법(Western blot)관찰소서시망막백세포개소(IL)-1β단백표체량;실시역전록-취합매련반응(RT-PCT)검측소서시망막IL-1β mRNA상대표체량.결과 시망막포편검사결과현시,OIR모형조시망막신생혈관면적교정상대조조명현증대,차이유통계학의의(t=15.02,P＜0.01);PEDF치료조시망막신생혈관면적교PBS간예대조조명현감소,차이유통계학의의(t=5.96,P＜0.01).형광현미경관찰결과현시,OIR모형조시망막가견외총상층(OPL)여신경절세포층(GCL)지간적신생혈관족교정상대조조명현증다;PEDF치료조시망막OPL여GCL지간적신생혈관족교PBS간예대조조명현감소.Western blot검측결과현시,OIR모형조시망막IL-1β단백표체수평교정상대조조현저제고,차이균유통계학의의(t=3.35,P＜0.05).PEDF치료조여PBS간예대조조비교,IL-1β단백표체수평하강,차이유통계학의의(P＜0.05).정상대조조여PEDF치료조비교,IL-1β단백표체수평무현저차이(F=11.764,P＞0.05).실시RT-PCR검측결과현시,OIR모형조시망막IL-1βmRNA상대표체량교정상대조조명현증가,차이유통계학의의(t=4.43,P＜0.01).PEDF치료조여PBS간예대조조비교,전자IL-1β mRNA상대표체량현저감소,차이유통계학의의;정상대조조여PEDF치료조비교,IL-1β mRNA상대표체량무현저차이(F=11.151,P＞0.05).결론 PEDF가억제OIR시망막신생혈관적형성.하조IL-1β재시망막적표체가능시기궤제지일.
Objective To study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice,and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF.Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group,oxygen-induced retinopathy (OIR) model group,PEDF treatment group and PBS treatment control group.All mice except normal control group with their mothers were exposed to (75 ± 2)％ oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model.Mice in normal control group were kept in room air only.At P12 and P14,respectively,mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl),while PBS treatment control group received the same volume of PBS (10 mmol/L,pH7.4).All mice were euthanized at P17 and eyes were isolated.The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy.Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR).Results Changes of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina,the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t =15.02,P＜0.01),and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96,P＜0.01).Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group.The specific expression levels of IL-1β protein in retinas of OIR mice by Western blot analysis were higher than those of normal control group(t=3.35,P＜0.05),While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P＜0.01),and there were no difference in normal control group and PEDF-treated group (F=11.764,P＞0.05).Similarly,expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t =4.43,P ＜ 0.01).After treated with PEDF,expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P ＜ 0.01),and there were no difference in normal control group and PEDF-treated group (F=11.15,P＞0.05).Conclusions PEDF can inhibit oxygen-induced retinal neovascularization.The mechanism may be related to that PEDF can downregulate the expression of IL 1β in retina.