VEGF小干扰RNA抑制兔角膜新生血管的实验研究
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摘要
研究背景:血管内皮生长因子(Vascular endothelial growthfactor,VEGF)是角膜新生血管形成最主要的生长因子,研究VEGF在角膜新生血管形成中的致病机制,调控其表达将是防治角膜新生血管性疾病的有效途径。
目的:本研究以兔角膜碱烧伤新生血管动物模型为研究对象,探讨VEGF小干扰RNA(VEGF siRNA)对碱烧伤后角膜组织中VEGFmRNA和蛋白质表达的影响,进而评价其在治疗角膜新生血管疾病中的作用。
方法:本研究分三部分进行。第一部分:取普通家兔25只,采用碱烧伤方法(1mol/L NaOH溶液)诱导角膜新生血管模型,碱烧伤后不同时间(1、3、5、7、14d)采用形态学方法观察角膜新生血管的生长情况,免疫组织化学方法检测角膜VEGF的表达。第二部分:25只普通家兔双眼角膜碱烧伤后(1mol/L NaOH溶液)立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGFsiRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为对照。碱烧伤后1、3、5、7、14 d,形态学分析评价角膜新生血管的生长情况。第三部分:在完成角膜形态学分析后,分别于碱烧伤后1、3、5、7、14d,采用RT-PCR方法检测角膜组织中VEGF mRNA的表达,免疫组织化学法检测角膜组织中VEGF蛋白质的表达。
结果:第一部分:角膜碱烧伤后24hVEGF的表达开始升高,伤后5d达高峰,伤后7dVEGF的表达开始下降,14d后显著下降。碱烧伤后,角膜新生血管与VEGF的表达呈明显的平行关系。第二部分:与对照眼相比,碱烧伤后球结膜下注射VEGFsiRNA重组质粒,实验眼在各时间段,CNV长度明显变短,面积明显变小,差异有统计学意义(p＜0.05)。第三部分:与对照眼相比较,实验组在各时间段RT-PCR结果显示角膜组织中VEGF mRNA的表达明显下调,免疫组织化学结果显示角膜组织中VEGF蛋白质的表达明显下调,差异有统计学意义(P＜0.05)。
结论:碱烧伤后兔角膜VEGF的表达水平与新生血管的形成有相关性,并在其中发挥了重要作用。VEGFsiRNA能有效地抑制碱烧伤后角膜组织中VEGF mRNA及蛋白质的表达,从而抑制角膜新生血管的形成。
Background: Vascular endothelial growth factor (VEGF) is the main growth factor which induced neovascularization in corneas. Investigating its mechanism in corneal neovascularization and regulating its expression will be the effective route of prerventing and healing corneal neovascularization diseases.
Purpose: To investigate the effect of VEGF small interefering RNA (VEGFsiRNA)on the expression of VEGFmRNA and protein in corneal tissues in rabbits after alkaline cauterization of the cornea and evaluate its role in curing corneal neovascularization diseases.
Methods:This study was divided into three parts. Part I: CNV was induced in vivo by alkaline cauterization (1mol/L NaOH)of the corneas in 25 rabbits. Cornea neovascularization were evaluated by morphometric analysis at different time points after alkaline burn (1, 3, 5, 7, 14d) . The expression of VEGF in corneas was detected by immunohistochemistry. Part II: CNV was induced in vivo by alkaline cauterization of the corneas in 25 rabbits. Using liposome (LF2000) as vector, VEGFsiRNA recombinant plasmid was injected into the subconjunctiva of the right eye ,and pSilencer2.1-U6 hygro was injected into the subconjunctiva of the left eye as control. 1 to 14 days post-cautery,CNV was evaluated with morphometric analysis. Part III: After the morphometric analysis , the expression of VEGFmRNA in rabbit corneas was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of VEGF protein in rabbit corneas was detected by immunohistochemical analysis 1, 3, 5, 7, 14 days after alkaline cautery of corneas.
Results: Part I: The expression of VEGF increased at 24 hours after cautery, peaded on 5d after cautery, decreased on 7d and significantly decreased to near baseline on 14d. After alkaline burn, the expression of VEGF paralleled to the growth of CNV in rabbit corneas. Part II: Comparing with the control eyes, the lenghth and area of CNV in the eyes treated with VEGFsiRNA recombinant plasmid at different time points after alkaline cauterization were shortened and reduced significantly (P<0.05) . Part III: Compared with the control eyes, the expression of VEGF mRNA and protein in corneas of eyes treated with VEGFsiRNA recombinant plasmid was significantly downregulated at different time points after alkaline cauterization (P<0.05 ) .
Conclusions: This investigation suggests that the expression of VEGF correlated with angiogenesis induced by alkaline cautery in the rabbit corneas and played an important role in rabbit corneal neovascularization. VEGFsiRNA can effectively inhibit the expression of VEGFmRNA and VEGF protein in corneas in rabbits after alkali burn, and suppress the development of CNV in rabbits after alkaline cauterization.
目的:本研究以兔角膜碱烧伤新生血管动物模型为研究对象,探讨VEGF小干扰RNA(VEGF siRNA)对碱烧伤后角膜组织中VEGFmRNA和蛋白质表达的影响,进而评价其在治疗角膜新生血管疾病中的作用。
方法:本研究分三部分进行。第一部分:取普通家兔25只,采用碱烧伤方法(1mol/L NaOH溶液)诱导角膜新生血管模型,碱烧伤后不同时间(1、3、5、7、14d)采用形态学方法观察角膜新生血管的生长情况,免疫组织化学方法检测角膜VEGF的表达。第二部分:25只普通家兔双眼角膜碱烧伤后(1mol/L NaOH溶液)立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGFsiRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为对照。碱烧伤后1、3、5、7、14 d,形态学分析评价角膜新生血管的生长情况。第三部分:在完成角膜形态学分析后,分别于碱烧伤后1、3、5、7、14d,采用RT-PCR方法检测角膜组织中VEGF mRNA的表达,免疫组织化学法检测角膜组织中VEGF蛋白质的表达。
结果:第一部分:角膜碱烧伤后24hVEGF的表达开始升高,伤后5d达高峰,伤后7dVEGF的表达开始下降,14d后显著下降。碱烧伤后,角膜新生血管与VEGF的表达呈明显的平行关系。第二部分:与对照眼相比,碱烧伤后球结膜下注射VEGFsiRNA重组质粒,实验眼在各时间段,CNV长度明显变短,面积明显变小,差异有统计学意义(p＜0.05)。第三部分:与对照眼相比较,实验组在各时间段RT-PCR结果显示角膜组织中VEGF mRNA的表达明显下调,免疫组织化学结果显示角膜组织中VEGF蛋白质的表达明显下调,差异有统计学意义(P＜0.05)。
结论:碱烧伤后兔角膜VEGF的表达水平与新生血管的形成有相关性,并在其中发挥了重要作用。VEGFsiRNA能有效地抑制碱烧伤后角膜组织中VEGF mRNA及蛋白质的表达,从而抑制角膜新生血管的形成。
Background: Vascular endothelial growth factor (VEGF) is the main growth factor which induced neovascularization in corneas. Investigating its mechanism in corneal neovascularization and regulating its expression will be the effective route of prerventing and healing corneal neovascularization diseases.
Purpose: To investigate the effect of VEGF small interefering RNA (VEGFsiRNA)on the expression of VEGFmRNA and protein in corneal tissues in rabbits after alkaline cauterization of the cornea and evaluate its role in curing corneal neovascularization diseases.
Methods:This study was divided into three parts. Part I: CNV was induced in vivo by alkaline cauterization (1mol/L NaOH)of the corneas in 25 rabbits. Cornea neovascularization were evaluated by morphometric analysis at different time points after alkaline burn (1, 3, 5, 7, 14d) . The expression of VEGF in corneas was detected by immunohistochemistry. Part II: CNV was induced in vivo by alkaline cauterization of the corneas in 25 rabbits. Using liposome (LF2000) as vector, VEGFsiRNA recombinant plasmid was injected into the subconjunctiva of the right eye ,and pSilencer2.1-U6 hygro was injected into the subconjunctiva of the left eye as control. 1 to 14 days post-cautery,CNV was evaluated with morphometric analysis. Part III: After the morphometric analysis , the expression of VEGFmRNA in rabbit corneas was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of VEGF protein in rabbit corneas was detected by immunohistochemical analysis 1, 3, 5, 7, 14 days after alkaline cautery of corneas.
Results: Part I: The expression of VEGF increased at 24 hours after cautery, peaded on 5d after cautery, decreased on 7d and significantly decreased to near baseline on 14d. After alkaline burn, the expression of VEGF paralleled to the growth of CNV in rabbit corneas. Part II: Comparing with the control eyes, the lenghth and area of CNV in the eyes treated with VEGFsiRNA recombinant plasmid at different time points after alkaline cauterization were shortened and reduced significantly (P<0.05) . Part III: Compared with the control eyes, the expression of VEGF mRNA and protein in corneas of eyes treated with VEGFsiRNA recombinant plasmid was significantly downregulated at different time points after alkaline cauterization (P<0.05 ) .
Conclusions: This investigation suggests that the expression of VEGF correlated with angiogenesis induced by alkaline cautery in the rabbit corneas and played an important role in rabbit corneal neovascularization. VEGFsiRNA can effectively inhibit the expression of VEGFmRNA and VEGF protein in corneas in rabbits after alkali burn, and suppress the development of CNV in rabbits after alkaline cauterization.
引文
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