中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2008年
10期
921-928
,共8页
蒋剑%夏晓波%许惠卓%熊宇%宋伟涛%熊思齐%李岩
蔣劍%夏曉波%許惠卓%熊宇%宋偉濤%熊思齊%李巖
장검%하효파%허혜탁%웅우%송위도%웅사제%리암
视网膜新生血管化%RNA,小分子干扰%核蛋白质类%DNA结合蛋白质类%血管内皮生长因子类%基因疗法
視網膜新生血管化%RNA,小分子榦擾%覈蛋白質類%DNA結閤蛋白質類%血管內皮生長因子類%基因療法
시망막신생혈관화%RNA,소분자간우%핵단백질류%DNA결합단백질류%혈관내피생장인자류%기인요법
Retinal neovascularization%RNA,small interfering%Nuclear proteins%DNA-binding proteins%Vascular endothelial growth factors%Gene therapy
目的 探讨缺氧诱导因子1α(HIF-1α)和血管内皮生长因子(VEGF)小片段干扰性RNA(siRNA)对小鼠视网膜新生血管的抑制作用.方法 本研究采用随机对照方法.构建HIF-1αsiRNA重组质粒.C57BL/6J小鼠玻璃体腔注射增强型绿色荧光蛋白(EGFP)表达质粒pEGFP-N1,1 d后视网膜铺片观察绿色荧光蛋白(GFP)的表达.选7天龄C57BL/6J小鼠90只,17只为正常组,73只建立氧诱导的视网膜新生血管模型,分为模型对照组、空载体组和基因治疗组(HIF-1α siRNA组、VEGF siRNA组及共转染组).于出氧舱前1 d,向空载体组小鼠玻璃体腔注射空载体质粒;HIF-1α siRNA组注射HIF-1α siRNA,VEGF siRNA组注射VEGF165 siRNA,共转染组注射HIF-1αsiRNA+VEGF165 siRNA.采用荧光造影视网膜铺片方法观察血管形态变化;制作组织切片计算突破视网膜内界膜的血管内皮细胞核数;采用逆转录聚合酶链反应和Western blot检测视网膜HIF-1α和VEGF的表达.采用单因素方差分析和组间最小显著差法进行统计学分析.结果 pEGFP-N1经脂质体LF2000介导转染视网膜细胞后1 d即可见GFP表达.基因治疗组较模型对照组新生血管丛明显减少,荧光渗漏明显减轻,其中共转染组效果最明显.基因治疗组[HIF-1α siRNA组为(27.73±2.33)个,VEGF siRNA组为(15.43±3.23)个,共转染组为(8.70 ±2.88)个]较其他3组突破视网膜内界膜的细胞核数量减少,差异均具有统计学意义(F=3016.527,P＜0.01).视网膜HIF-1α mRNA及蛋白表达水平:模型对照组(1.08±0.06,0.383±0.009)和空载体组(1.09±0.05,0.386 ±0.010)较正常组(0.81 ±0.07,0.035±0.003)上调,而HIF-1α siRNA组(0.46±0.06,0.182±0.008)较模型对照组下调,抑制效率分别为57.4%和52.5%,差异均有统计学意义(F=139.804,2686.001;P＜0.01).VEGF mRNA及蛋白表达水平:模型对照组(1.53 ±0.07,0.340±0.004)和空载体组(1.59±0.06,0.337±0.009)较正常组(0.27±0.08,0,051±0.008)明显上调,而基因治疗组较模型对照组明显下调,差异均有统计学意义(F=421.423,2513.583;P＜0.01),其中共转染组下调最明显,抑制效率分别为85.6%和80.9%.结论 HIF-1α siRNA和VEGF165siRNA均可有效抑制小鼠视网膜新生血管形成,两种siRNA共转染抑制效果最明显.
目的 探討缺氧誘導因子1α(HIF-1α)和血管內皮生長因子(VEGF)小片段榦擾性RNA(siRNA)對小鼠視網膜新生血管的抑製作用.方法 本研究採用隨機對照方法.構建HIF-1αsiRNA重組質粒.C57BL/6J小鼠玻璃體腔註射增彊型綠色熒光蛋白(EGFP)錶達質粒pEGFP-N1,1 d後視網膜鋪片觀察綠色熒光蛋白(GFP)的錶達.選7天齡C57BL/6J小鼠90隻,17隻為正常組,73隻建立氧誘導的視網膜新生血管模型,分為模型對照組、空載體組和基因治療組(HIF-1α siRNA組、VEGF siRNA組及共轉染組).于齣氧艙前1 d,嚮空載體組小鼠玻璃體腔註射空載體質粒;HIF-1α siRNA組註射HIF-1α siRNA,VEGF siRNA組註射VEGF165 siRNA,共轉染組註射HIF-1αsiRNA+VEGF165 siRNA.採用熒光造影視網膜鋪片方法觀察血管形態變化;製作組織切片計算突破視網膜內界膜的血管內皮細胞覈數;採用逆轉錄聚閤酶鏈反應和Western blot檢測視網膜HIF-1α和VEGF的錶達.採用單因素方差分析和組間最小顯著差法進行統計學分析.結果 pEGFP-N1經脂質體LF2000介導轉染視網膜細胞後1 d即可見GFP錶達.基因治療組較模型對照組新生血管叢明顯減少,熒光滲漏明顯減輕,其中共轉染組效果最明顯.基因治療組[HIF-1α siRNA組為(27.73±2.33)箇,VEGF siRNA組為(15.43±3.23)箇,共轉染組為(8.70 ±2.88)箇]較其他3組突破視網膜內界膜的細胞覈數量減少,差異均具有統計學意義(F=3016.527,P＜0.01).視網膜HIF-1α mRNA及蛋白錶達水平:模型對照組(1.08±0.06,0.383±0.009)和空載體組(1.09±0.05,0.386 ±0.010)較正常組(0.81 ±0.07,0.035±0.003)上調,而HIF-1α siRNA組(0.46±0.06,0.182±0.008)較模型對照組下調,抑製效率分彆為57.4%和52.5%,差異均有統計學意義(F=139.804,2686.001;P＜0.01).VEGF mRNA及蛋白錶達水平:模型對照組(1.53 ±0.07,0.340±0.004)和空載體組(1.59±0.06,0.337±0.009)較正常組(0.27±0.08,0,051±0.008)明顯上調,而基因治療組較模型對照組明顯下調,差異均有統計學意義(F=421.423,2513.583;P＜0.01),其中共轉染組下調最明顯,抑製效率分彆為85.6%和80.9%.結論 HIF-1α siRNA和VEGF165siRNA均可有效抑製小鼠視網膜新生血管形成,兩種siRNA共轉染抑製效果最明顯.
목적 탐토결양유도인자1α(HIF-1α)화혈관내피생장인자(VEGF)소편단간우성RNA(siRNA)대소서시망막신생혈관적억제작용.방법 본연구채용수궤대조방법.구건HIF-1αsiRNA중조질립.C57BL/6J소서파리체강주사증강형록색형광단백(EGFP)표체질립pEGFP-N1,1 d후시망막포편관찰록색형광단백(GFP)적표체.선7천령C57BL/6J소서90지,17지위정상조,73지건립양유도적시망막신생혈관모형,분위모형대조조、공재체조화기인치료조(HIF-1α siRNA조、VEGF siRNA조급공전염조).우출양창전1 d,향공재체조소서파리체강주사공재체질립;HIF-1α siRNA조주사HIF-1α siRNA,VEGF siRNA조주사VEGF165 siRNA,공전염조주사HIF-1αsiRNA+VEGF165 siRNA.채용형광조영시망막포편방법관찰혈관형태변화;제작조직절편계산돌파시망막내계막적혈관내피세포핵수;채용역전록취합매련반응화Western blot검측시망막HIF-1α화VEGF적표체.채용단인소방차분석화조간최소현저차법진행통계학분석.결과 pEGFP-N1경지질체LF2000개도전염시망막세포후1 d즉가견GFP표체.기인치료조교모형대조조신생혈관총명현감소,형광삼루명현감경,기중공전염조효과최명현.기인치료조[HIF-1α siRNA조위(27.73±2.33)개,VEGF siRNA조위(15.43±3.23)개,공전염조위(8.70 ±2.88)개]교기타3조돌파시망막내계막적세포핵수량감소,차이균구유통계학의의(F=3016.527,P＜0.01).시망막HIF-1α mRNA급단백표체수평:모형대조조(1.08±0.06,0.383±0.009)화공재체조(1.09±0.05,0.386 ±0.010)교정상조(0.81 ±0.07,0.035±0.003)상조,이HIF-1α siRNA조(0.46±0.06,0.182±0.008)교모형대조조하조,억제효솔분별위57.4%화52.5%,차이균유통계학의의(F=139.804,2686.001;P＜0.01).VEGF mRNA급단백표체수평:모형대조조(1.53 ±0.07,0.340±0.004)화공재체조(1.59±0.06,0.337±0.009)교정상조(0.27±0.08,0,051±0.008)명현상조,이기인치료조교모형대조조명현하조,차이균유통계학의의(F=421.423,2513.583;P＜0.01),기중공전염조하조최명현,억제효솔분별위85.6%화80.9%.결론 HIF-1α siRNA화VEGF165siRNA균가유효억제소서시망막신생혈관형성,량충siRNA공전염억제효과최명현.
Objective To evaluate the inhibitory effect of small interfering RNA(siRNA)on the expressions of hypoxia inducible factor-1α(HiF-1α)and vascular endothelial gowth factor(VEGF)in retinal neovascularization in the mouse.Methods HIF-1α siRNA recombinant plasmid was constructed.Liposome mediated the expressive plasmid of enhanced green fluorescent protein(EGFP)pEGFP-N1 complex was injected into the vitreous of C57BL/6J mice.The expression of GFP wag observed in retinal flat-mounts one day after injection.Randomized controlled trial was performed. There were totally ninety(seven-day-old)C57BL/6J mice in which seventeen mice were chosen as normal group and seventy-three mice were divided into five groups randomly including control model group,vector group and gene therapy group(HIF-1α siRNA group,VEGF siRNA group and co-transfection group),in which retinal neovascularization was induced by hypoxia.Liposome with vector plasmid,HIF-1αt siRNA and VEGF165 siRNA were injected into the vitreous in the vector group,HIF-1α siRNA group and VEGF siRNA group respectively one day before mice were moved out to room air from the cabin.Liposome with HIF-1α siRNA and VEGF165 siRNA was injected in the co-transfection group at the same time point.Fluorescent angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections.HIF-1α and VEGF levels in retinas were measured by reverse transcriptase-polymerase chain reaction and Western blot.Significant differences between groups were evaluated bv one-way analysis of variance,followed by a least-significant difference analysis.Results The GFP expression in retinal cells was observed one day after injection of liposome mediated pEGFP-N1 complex.Neovascular tufts and fluorescein leakage were decreased in gene therapy group especially in co-transfection group compared to the control model group.The neovascular nuclei were decreased in gene therapy group[HIF-1α siRNA group(27.73±2.33),VEGF siRNA group(15.43±3.23),co-transfection group(8.70±2.88)]compared to the other three groups(F=3016.537,P＜0.01).The expression of HIF-1α mRNA and protein in retinas were increased in control model group (1.08±0.06,0.383±0.009)and vector group(1.09±0.05,0.386±0.010)as compared with normal group(0.81 ±0.07,0.035 ±0.003),while decreased 57.4%and 52.5%respectively in the HIF-1α siRNA group(0.46±0.06,0.182±0.008)as compared with control model group(F=139.804,2686.001;P＜0.01).The expression of VEGF mRNA and protein in retinas were increased significantly in control model group(1.53±0.07,0.340±0.004)and vector group(1.59±0.06,0.337 ±0.009)as compared with normal group(0.27±0.08,0.051±0.008),while decreased significantly in gene therapy group especially co-transfection group(decreased 85.6% and 80.9%respectively)as compared with control model group(F=421.423,2513.583;P＜0.01).Conclusions HIF-1α siRNA and VEGF165 siRNA can inhibit retinal neovascularization in the mouse effectively.Co-transfection of these two siRNAs shows the greatest inhibitory effect.
