miRNA-410对小鼠视网膜新生血管抑制作用的研究
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摘要
视网膜新生血管性疾病，如糖尿病性视网膜病变、视网膜静脉阻塞和早产儿视网膜病变等均会导致视网膜新生血管化。目前视网膜新生血管性疾病，是世界范围内最严重的致盲性眼病之一，在发达国家已上升为致盲性眼病的首要原因。随着我国人民生活水平提高，眼底新生血管性疾病也日渐成为威胁我国人民视力健康的重要原因。视网膜新生血管的形成可引起玻璃体出血、牵拉性视网膜脱离和新生血管性青光眼，其发病机制并未完全清楚。但近年来生长因子在视网膜新生血管形成中的作用已形成共识。局部组织新生血管刺激因子和抑制因子动态平衡的失调是产生新生血管的关键。已发现的刺激血管生长的因子包括：血管内皮生长因子(vascular endothelial growthfactor，VEGF)、成纤维细胞生长因子(fibroblast growth factor，FGF)、白细胞介素8(interlukin-8)等。已发现的抑制血管生长的因子有转化生长因子(transforming growthfactor beta，TGF-β)和血栓素(thrombospondin)等。VEGF是视网膜新生血管形成最主要的生长因子。虽然视网膜新生血管形成的机制尚未完全明了，许多调节因素也仍然不清，但是许多研究表明，抑制VEGF的表达，可以减少视网膜新生血管的形成。
microRNA (miRNA)是一种长度约22nt的内源性，非编码单链小分子RNA，与靶基因mRNA的3′非翻译区进行碱基互补配对后，发挥对mRNA的降解作用或抑制mRNA的翻译，从而对基因进行转录后表达的调控。大约30%的蛋白质编码基因是由miRNA调控的。miRNA在细胞的增殖，分化和凋亡以及所报道的所有生物学进程中都发挥重要的作用。microRNA已经在肿瘤、心血管等方面研究取得了一定的进展，其在眼科的应用也引起了学者们的兴趣，有研究发现在视网膜和眼部其他组织中，microRNA的表达具有组织特异性和发育阶段特异性，提示其在视网膜和眼部其他组织中具有潜在的组织和细胞的功能特异性。
本实验通过构建小鼠microRNA-410质粒载体，以高氧诱导的小鼠视网膜新生血管模型为实验对象，将构建好的浓缩质粒通过以下两种不同的方式：1.玻璃体腔注射浓缩质粒入小鼠眼球，5天后取材观察疗效。2.将浓缩质粒制成眼药水，每天对实验动物局部点眼药水观察治疗效果，持续5天。最后研究结果表明microRNA-410可通过下调VEGF的表达抑制血管内皮细胞的增殖，减少视网膜新生血管数量，从而阻碍新生血管的形成。
第一部分miRNA-410质粒载体的构建及验证
目的构建miRNA-410质粒载体，并在细胞水平验证其对VEGF的抑制作用
方法构建miRNA-410重组表达质粒（pLKO.1-miRNA-410）；采用脂质体法分别将表达质粒和空质粒转入人脐静脉内皮细胞(HUVEC)。转染后采用RT-PCR法检测转染后重组质粒的表达情况；实时定量PCR及western blot检测VEGF表达的改变。
结果成功构建了miRNA-410重组质粒。转染了pLKO.1-miRNA-410的HUVEC中VEGF表达较转染pLKO.1-mock中的表达明显下调(p<0.05),转染了pLKO.1-miRNA-410-antisense的HUVEC中VEGF表达较转染pLKO.1-mock中的表达明显上调(p<0.05)。
结论成功构建了miRNA-410重组质粒，并在细胞水平验证其对VEGF的抑制作用。
第二部分可定量的氧诱导小鼠视网膜新生血管模型的建立及VEGFmRNA的动态表达
目的建立可对视网膜新生血管进行定量研究的动物模型，为下一步研究视网膜新生血管发生机制及治疗提供实验基础。
方法选择健康7日龄(P7)C57BL/6J小鼠，雌雄不限。共16窝100只，随机分为高氧组(OIR组)和对照组(C组)。高氧组：将50只7日龄的C57BL／6J小鼠（P7）连同母鼠放在浓度为(75±2)％的氧浓度环境中，5天后回到正常空气中，作为氧诱导模型组；对照组：另50只同日龄新生小鼠置于正常空气环境中生活，作为正常对照组。两组小鼠分别在P13、P15、P17、P19、P23分批处死，摘除眼球，通过FITC-Dextran荧光造影视网膜铺片观察视网膜新生血管形态变化；组织切片观察并计数矢状面5mm视网膜切片中突破内界膜的血管内皮细胞核数，反映视网膜血管的增生情况；real-time RT-PCR检测视网膜中VEGFmRNA的动态表达。
结果荧光素血管灌注法视网膜铺片结果：正常组小鼠视网膜血管分布呈均匀的网状，未见新生血管和无灌注区。高氧诱导组小鼠回到正常空气环境1天（P13）后，视网膜开始出现新生血管；到空气中3天（P15）后，视网膜新生血管增多明显，到空气中5天（P17）后，视网膜新生血管形成达到高峰，到空气中7天（P19）后，视网膜新生血管逐渐减少，到空气中12天(P23)后，视网膜新生血管消退。视网膜切片结果：突破视网膜内界膜的血管内皮细胞数，在P13.P15.P17.P19.P23氧诱导的模型组分别为(1.39±1.12个)、（16.67±3.51个）、（42.31±4.69个）、（39.23±4.5个）、（0.93±0.85个），正常对照组分别为（0.96±0.91个）、（0.94±0.92个）、(0.89±0.91个)、（0.9±0.89个）、（0.89±0.97个），二者相比差异有显著意义(p<0.05)。高氧诱导模型组各时间点视网膜组织中的VEGFmRNA表达水平较同时间点对照组明显上调，二者相比差异有显著意义(p<0.01)。
结论该动物模型成模率高，可重复性好，并可进行定量研究，是进行小鼠视网膜新生血管发生机制和药物治疗研究的合适模型。RT-PCR结果显示在氧诱导增殖性视网膜病变小鼠视网膜组织中VEGFmRNA表达明显上调，其变化趋势与视网膜新生血管形成相对应。
第三部分miRNA-410抑制小鼠视网膜新生血管形成的研究
目的观察miRNA-410对视网膜新生血管形成的抑制作用，进一步阐明miRNA-410抑制视网膜新生血管形成中的调控作用。
方法选择健康7日龄(P7)C57BL/6J小鼠，雌雄不限。共10窝共60只C57BL／6J小鼠。随机取10只在正常氧环境下饲养的C57BL／6J小鼠为正常对照组。剩余50只鼠龄为7天的C57BL／6J小鼠置于浓度为(75±2)％高氧环境中生活5天，在P12返回正常氧环境中。在高氧处理过的50只小鼠中，随机取其中10只，不做任何药物处理，作为高氧诱导模型组。剩余40只从高氧环境中取出的小鼠，随机取10只小鼠于出氧舱后每日右眼局部点pLKO.1-miRNA-410眼药水，2/日,作为pLKO.1-miRNA-410眼药水组。随机取10只小鼠于出氧舱后每日右眼局部点pLKO.1-mock眼药水，2/日，作为pLKO.1-mock眼药水组。10只小鼠于出氧舱后当日右眼玻璃体腔内注射0.4μlpLKO.1-miRNA-410，作为眼内注射pLKO.1-miRNA-410组，10只小鼠于出氧舱后当日右眼玻璃体腔内注射0.4μl pLKO.1-mock，作为眼内注射pLKO.1-mock组。以上5组小鼠的左眼均不做外源性处理。所有小鼠在P17时，在麻醉状态下取材，组织学切片观察突破视网膜内界膜的血管内皮细胞核数量；FITC-Dextran荧光造影视网膜铺片了解视网膜血管形态的改变；RT-PCR检测视网膜组织中VEGFmRNA表达水平。
结果组织学切片结果表明：高氧诱导组突破视网膜内界膜的血管内皮细胞核数与正常组比较差异有显著性(p<0.05)；局部使用pLKO.1-miRNA-410眼药水组和玻璃体腔注射pLKO.1-miRNA-410组与高氧诱导组比较差异有显著性(p<0.05)，与正常组比较差异无显著性(p＞0.05)；局部使用pLKO.1-miRNA-410眼药水组与局部使用pLKO.1-miRNA-mock眼药水组比较差异有显著性(p<0.05)；玻璃体腔注射pLKO.1-miRNA-410组与玻璃体腔注射pLKO.1-miRNA-mock组比较差异有显著性(p<0.05)；局部使用pLKO.1-miRNA-410眼药水组和玻璃体腔注射pLKO.1-miRNA-410组与高氧诱导组比较差异无显著性(p＞0.05)
视网膜铺片结果显示：玻璃体腔注射pLKO.1-miRNA-410组较高氧诱导模型组、玻璃体腔内注射pLKO.1-mock组新生血管丛明显减少，渗漏明显减轻；局部使用pLKO.1-miRNA-410眼药水组较高氧诱导模型组、玻璃体腔注射pLKO.1-miRNA-410组、玻璃体腔内注射pLKO.1-mock组和局部pLKO.1-mock眼药水组新生血管丛明显减少，荧光渗漏最轻。
视网膜组织VEGFmRNA水平检测：1.玻璃体腔注射pLKO.1-miRNA-410组与玻璃体腔注射pLKO.1-mock相比，VEGFmRNA表达降低。差异有显著性（p<0.05）2.局部使用pLKO.1-miRNA-410眼药水组与局部使用pLKO.1-mock眼药水组相比，VEGFmRNA表达降低。差异有显著性（p<0.05）3.局部使用pLKO.1-miRNA-410眼药水组与玻璃体腔注射pLKO.1-miRNA-410组相比，VEGFmRNA表达降低。差异无显著性（p＞0.05）
结论miRNA-410可有效抑制视网膜新生血管的形成，进一步证明了miRNA-410在视网膜新生血管形成中所起的调控作用,同时为血管增生性视网膜病变的治疗提供了新的途径。
The retinal neovascular diseases, such as diabetic retinopathy, retinal vein occlusionand retinopathy of prematurity, can cause retinal neovascularization (RNV). Disease ofretinal ncovascular is one of the most serious diseases which can cause blindness in humanbeings all around the world. In developed country, the retinal neovascular disease is theleading cause of blindless. With the improved living standards, the retinal neovasculardisease has also become a threat to the visual health of our people. Retinalneovascularization can lead to vitreous hemorrhage，traction retinal detachment andneovascular glaucoma，even blindness, but the accurate patholgenesis is still unknown. Inrecent years, the roles of growth factors in the pathogenesis of RNV come to be theconsensus for most experts.
The imbalance of the neovascularization stimulating factor and inhibitory factor inlocal tissue play a vital role in producing new blood vessels. These stimulate blood vesselgrowth factors include vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF) and interlukin-8. The Inhibition of vascular endothelial growth factor includetransforming growth factor beta (TGF-β) and thrombospondin. VEGF is the main growthfactor which induced neovascularization in retina. We can reduce the formation of retinalneovascularization through inhibiting the expression of VEGF.
microRNAs (miRNAs) are endogenous, small, non-coding, regulatory RNAs,-22nucleotides (nts) in size. miRNAs target their downstream messenger RNAs by basepairing to their target sites with sequence complementarity, mainly in the3′untranslatedregion (UTR), and induce the breakdown of the targeted mRNAs and/or inhibition oftranslation from the mRNAs. There are studies shown that microRNAs play importantroles in cellular proliferation, differentiation, and cell death. microRNA involved in aseries of life processes through regulating gene expression. Approximately30%of theprotein-coding genes are estimated to be regulated by miRNAs. There are some progress inthe research of microRNA on oncology and cardiovascular. The scientists ofophthalmology are also attracted to the study of microRNA. Many miRNAs showedunique tissue-specific and developmental stage-specific expression patterns, suggestingpotential unique functions in the retina and other ocular tissues.
In this study,we aim to construct microRNA-410plasmid and investigate the effect ofmicroRNA-410on the inhibition of retinal neovascularization in mice of oxygen inducedretinopathy (OIR) and evaluate the efficiency of microRNA-410in retinal neovasculardisease．We use the condensed plasmid in two different ways. Firstly, intravitrealinjection was performed by inserting a needle into the vitreous of anesthetized OIR mice.Secondly, we make the condensed plasmid as the eye drops, drop it daily in the mouse eyesand observe the treatment effect. We found that microRNA-410inhibited the of retinalneovascularization in mice of OIR. The resultsshowed that miR-410reduced retinal NV inOIR model. The expression of VEGF was significantly reduced in the OIR model whichwas treated with miR-410, suggesting that miR-410may play a potential role in thepathogenesis in oxygen induced retinopathy.
Chapter one：Construction and Verification of miRNA-410Plasmid vector
Objective To construct miRNA-410Plasmid vector of mouse and verify theinhibition of vascular endothelial growth factor (VEGF) at the cellular level.
Methods miRNA-410recombinant plasmid was construct. Transfect themiRNA-410recombinant plasmids (cotransfection group) by the Lipofectamine2000method. Detect the expression of recombinant plasmid after transfection by Real-timePCR.Use RT-PCR to detect the expression change of VEGF in the Human Umbilical VeinEndothelial Cells（HUVEC）.
Results We constructed the miRNA-410recombinant plasmid(pLKO.1-miRNA-410) successfully. The expression of recombinant plasmids wereidentified by RT-PCR after24hours of transfection to the HUVEC. The level of VEGF ofthe pLKO.1-miRNA-410group is significantly downregulated than the pLKO.1-mock(p<0.05) in HUVEC. The level of VEGF of the pLKO.1-miRNA-410-antisense group issignificantly upregulated than the pLKO.1-mock group (p<0.05) in HUVEC.
Conclusions We constructed the miRNA-410recombinant plasmidsuccessfully and verify the inhibition of VEGF at the cellular level.
Chapter two： Establishment of a quantifying animal model ofretinal neovascularization and dynamic expression of VEGF
Objective To establish a quantifying model of retinal neovascularization suitablefor the study of pathogenesis and therapeutic intervention for the retinalneovascularization．
Methods One hundred7-day-old C57BL／6J mice were divided intooxygen-induced retinopathy group and control group．In oxygen-induced retinopathygroup，50mice were exposed to (75土2)％oxygen for5days and then to room air；incontrol group，50mice were raised in room air．Fourty mice of postnatal day13(P13),P15, P17, P19, P23were perfused and eyes were enucleated．The retinal preparationperfused with fluorescein-Dextran was examined to assess the oxygen-induced changes ofretinal vessels．The proliferated neovascular response was quantitated by counting thenuclei of endothelial cells of new vessels extending from the retina into the vitreous.Thedynamic expression of VEGF in the retinal was detected by real-time RT-PCR andImmunohistochemical detection.
Results The neovascularization in the eyes of oxygen-induced retinalpathy inmurine model was significantly increased compared with the normal group．There aremore construction and occlusion in retinal blood vessels and appears large tracts ofnon-perfused area.Similarly， histological analysis indicates that neovascular nucleiprotruding intoviteous cavity was increased compared to the normal group． Theneovascular nuclei protruding intoviteous cavity of oxygen-induced grouped is(1.39±1.12)，(16.67±3.51)，(42.31±4.69)，(39.23±4.5)，(0.93±0.85) in P13，P15，P17，P19，P23respectively，compared to normal group with value of (0.96±0.91)，(0.94±0.92),，(0.89±0.91)，(0.9±0.89)，(0.89±0.97) respectively.The expression of VEGFin oxygen-induced retinopathy group was dramatically increased than normal group.Thedifference was statistically significance (p<0.05).
Conclusions The reproducible and quantifiable mouse model of retinalneovascularization isuseful for the study of pathogenesis of retinal neovascularization andtherapeuticintervention．The expression of VEGFmRNA in mice with oxygen-inducedpreoliferative retinopathy is related to retinal anigogenesis.
Chapter three： Suppression of retinal neovascularization bymiRNA-410in Establishment of a quantifying animal model
Objective To evaluate the inhibitory effects of miRNA-410on retinalneovascularization in the mouse．
Methods Sixty7-day-old C57BL／6J mice were divided into oxygen-inducedretinopathy group and normal group．Ten Seven-day-old C57BL／6J mice were exposedto normal air randomly, fifty Seven-day-old C57BL／6J mice were exposed to (75±2)％oxygen for5days， then they were returned to the room air to induce retinalneovascularization．Ten of the oxygen-induced models were untreated.Ten of theoxygen-induced models were treated with eyedrops contains pLKO.1-miRNA-410.Ten ofthe oxygen-induced models were treated with eyedrops contains pLKO.1-mock.Ten ofthe oxygen-induced models were intrvitreally injected with pLKO.1-miRNA-410.Ten ofthe oxygen-induced models were intrvitreally injected with pLKO.1-mock. Retinalneovascularization was evaluated by angiography with injection of fluorescein dextran andthe proliferated neovascular response was quantitated by counting the nuclei of endothelialcells of new vessels extending from the retina into the vitreous.Moreover，RT-PCR wasused to detect the expression of VEGFmRNA in the retinas．
Results The neovascularization in the eyes of pLKO.1-miRNA-410intrvitrealinjection and pLKO.1-miRNA-410eyedrops group was significantly decreased comparedwith the oxygen-induced group．There are less construction and occlusion in retinal bloodvessels and appears smaller tracts of non-perfused area in this two groups. The expressionof VEGFmRNA in the two groups were dramatically decreased than that of theoxygen-induced group.The difference was statistically significance (p<0.05). Similarly， histological analysis indicates that neovascular nuclei protruding intoviteous cavity wasdecreased compared to the oxygen-induced group．
The neovascularization in the eyes of pLKO.1-miRNA-mock intrvitreal injection andpLKO.1-miRNA-mock eyedrops group was similar to the oxygen-induced group．Thereare many occlusions in retinal blood vessels and appears lage tracts of non-perfused area inthis two groups. The expression of VEGFmRNA in the two groups were dramaticallyincreased than that of the normal group.The difference was statistically significance(p<0.05). Similarly,histological analysis indicates that neovascular nuclei protrudingintoviteous cavity was increased compared to the normal group．The difference wasstatistically significance (p<0.05).
Conclusions Retinal neovascularization can be inhibited by intravitreal injectionof pLKO.1-miRNA-410,which suggests that intravitreal injection of pLKO.1-miRNA-410may have potential therapeutic benefits in ischemic-induced retinal diseases. miRNA-410can reduce the expression of VEGF in the retinas of mice．It may provide a powerful andnovel therapeutic tool for ischemic-induced retinal diseases．
microRNA (miRNA)是一种长度约22nt的内源性，非编码单链小分子RNA，与靶基因mRNA的3′非翻译区进行碱基互补配对后，发挥对mRNA的降解作用或抑制mRNA的翻译，从而对基因进行转录后表达的调控。大约30%的蛋白质编码基因是由miRNA调控的。miRNA在细胞的增殖，分化和凋亡以及所报道的所有生物学进程中都发挥重要的作用。microRNA已经在肿瘤、心血管等方面研究取得了一定的进展，其在眼科的应用也引起了学者们的兴趣，有研究发现在视网膜和眼部其他组织中，microRNA的表达具有组织特异性和发育阶段特异性，提示其在视网膜和眼部其他组织中具有潜在的组织和细胞的功能特异性。
本实验通过构建小鼠microRNA-410质粒载体，以高氧诱导的小鼠视网膜新生血管模型为实验对象，将构建好的浓缩质粒通过以下两种不同的方式：1.玻璃体腔注射浓缩质粒入小鼠眼球，5天后取材观察疗效。2.将浓缩质粒制成眼药水，每天对实验动物局部点眼药水观察治疗效果，持续5天。最后研究结果表明microRNA-410可通过下调VEGF的表达抑制血管内皮细胞的增殖，减少视网膜新生血管数量，从而阻碍新生血管的形成。
第一部分miRNA-410质粒载体的构建及验证
目的构建miRNA-410质粒载体，并在细胞水平验证其对VEGF的抑制作用
方法构建miRNA-410重组表达质粒（pLKO.1-miRNA-410）；采用脂质体法分别将表达质粒和空质粒转入人脐静脉内皮细胞(HUVEC)。转染后采用RT-PCR法检测转染后重组质粒的表达情况；实时定量PCR及western blot检测VEGF表达的改变。
结果成功构建了miRNA-410重组质粒。转染了pLKO.1-miRNA-410的HUVEC中VEGF表达较转染pLKO.1-mock中的表达明显下调(p<0.05),转染了pLKO.1-miRNA-410-antisense的HUVEC中VEGF表达较转染pLKO.1-mock中的表达明显上调(p<0.05)。
结论成功构建了miRNA-410重组质粒，并在细胞水平验证其对VEGF的抑制作用。
第二部分可定量的氧诱导小鼠视网膜新生血管模型的建立及VEGFmRNA的动态表达
目的建立可对视网膜新生血管进行定量研究的动物模型，为下一步研究视网膜新生血管发生机制及治疗提供实验基础。
方法选择健康7日龄(P7)C57BL/6J小鼠，雌雄不限。共16窝100只，随机分为高氧组(OIR组)和对照组(C组)。高氧组：将50只7日龄的C57BL／6J小鼠（P7）连同母鼠放在浓度为(75±2)％的氧浓度环境中，5天后回到正常空气中，作为氧诱导模型组；对照组：另50只同日龄新生小鼠置于正常空气环境中生活，作为正常对照组。两组小鼠分别在P13、P15、P17、P19、P23分批处死，摘除眼球，通过FITC-Dextran荧光造影视网膜铺片观察视网膜新生血管形态变化；组织切片观察并计数矢状面5mm视网膜切片中突破内界膜的血管内皮细胞核数，反映视网膜血管的增生情况；real-time RT-PCR检测视网膜中VEGFmRNA的动态表达。
结果荧光素血管灌注法视网膜铺片结果：正常组小鼠视网膜血管分布呈均匀的网状，未见新生血管和无灌注区。高氧诱导组小鼠回到正常空气环境1天（P13）后，视网膜开始出现新生血管；到空气中3天（P15）后，视网膜新生血管增多明显，到空气中5天（P17）后，视网膜新生血管形成达到高峰，到空气中7天（P19）后，视网膜新生血管逐渐减少，到空气中12天(P23)后，视网膜新生血管消退。视网膜切片结果：突破视网膜内界膜的血管内皮细胞数，在P13.P15.P17.P19.P23氧诱导的模型组分别为(1.39±1.12个)、（16.67±3.51个）、（42.31±4.69个）、（39.23±4.5个）、（0.93±0.85个），正常对照组分别为（0.96±0.91个）、（0.94±0.92个）、(0.89±0.91个)、（0.9±0.89个）、（0.89±0.97个），二者相比差异有显著意义(p<0.05)。高氧诱导模型组各时间点视网膜组织中的VEGFmRNA表达水平较同时间点对照组明显上调，二者相比差异有显著意义(p<0.01)。
结论该动物模型成模率高，可重复性好，并可进行定量研究，是进行小鼠视网膜新生血管发生机制和药物治疗研究的合适模型。RT-PCR结果显示在氧诱导增殖性视网膜病变小鼠视网膜组织中VEGFmRNA表达明显上调，其变化趋势与视网膜新生血管形成相对应。
第三部分miRNA-410抑制小鼠视网膜新生血管形成的研究
目的观察miRNA-410对视网膜新生血管形成的抑制作用，进一步阐明miRNA-410抑制视网膜新生血管形成中的调控作用。
方法选择健康7日龄(P7)C57BL/6J小鼠，雌雄不限。共10窝共60只C57BL／6J小鼠。随机取10只在正常氧环境下饲养的C57BL／6J小鼠为正常对照组。剩余50只鼠龄为7天的C57BL／6J小鼠置于浓度为(75±2)％高氧环境中生活5天，在P12返回正常氧环境中。在高氧处理过的50只小鼠中，随机取其中10只，不做任何药物处理，作为高氧诱导模型组。剩余40只从高氧环境中取出的小鼠，随机取10只小鼠于出氧舱后每日右眼局部点pLKO.1-miRNA-410眼药水，2/日,作为pLKO.1-miRNA-410眼药水组。随机取10只小鼠于出氧舱后每日右眼局部点pLKO.1-mock眼药水，2/日，作为pLKO.1-mock眼药水组。10只小鼠于出氧舱后当日右眼玻璃体腔内注射0.4μlpLKO.1-miRNA-410，作为眼内注射pLKO.1-miRNA-410组，10只小鼠于出氧舱后当日右眼玻璃体腔内注射0.4μl pLKO.1-mock，作为眼内注射pLKO.1-mock组。以上5组小鼠的左眼均不做外源性处理。所有小鼠在P17时，在麻醉状态下取材，组织学切片观察突破视网膜内界膜的血管内皮细胞核数量；FITC-Dextran荧光造影视网膜铺片了解视网膜血管形态的改变；RT-PCR检测视网膜组织中VEGFmRNA表达水平。
结果组织学切片结果表明：高氧诱导组突破视网膜内界膜的血管内皮细胞核数与正常组比较差异有显著性(p<0.05)；局部使用pLKO.1-miRNA-410眼药水组和玻璃体腔注射pLKO.1-miRNA-410组与高氧诱导组比较差异有显著性(p<0.05)，与正常组比较差异无显著性(p＞0.05)；局部使用pLKO.1-miRNA-410眼药水组与局部使用pLKO.1-miRNA-mock眼药水组比较差异有显著性(p<0.05)；玻璃体腔注射pLKO.1-miRNA-410组与玻璃体腔注射pLKO.1-miRNA-mock组比较差异有显著性(p<0.05)；局部使用pLKO.1-miRNA-410眼药水组和玻璃体腔注射pLKO.1-miRNA-410组与高氧诱导组比较差异无显著性(p＞0.05)
视网膜铺片结果显示：玻璃体腔注射pLKO.1-miRNA-410组较高氧诱导模型组、玻璃体腔内注射pLKO.1-mock组新生血管丛明显减少，渗漏明显减轻；局部使用pLKO.1-miRNA-410眼药水组较高氧诱导模型组、玻璃体腔注射pLKO.1-miRNA-410组、玻璃体腔内注射pLKO.1-mock组和局部pLKO.1-mock眼药水组新生血管丛明显减少，荧光渗漏最轻。
视网膜组织VEGFmRNA水平检测：1.玻璃体腔注射pLKO.1-miRNA-410组与玻璃体腔注射pLKO.1-mock相比，VEGFmRNA表达降低。差异有显著性（p<0.05）2.局部使用pLKO.1-miRNA-410眼药水组与局部使用pLKO.1-mock眼药水组相比，VEGFmRNA表达降低。差异有显著性（p<0.05）3.局部使用pLKO.1-miRNA-410眼药水组与玻璃体腔注射pLKO.1-miRNA-410组相比，VEGFmRNA表达降低。差异无显著性（p＞0.05）
结论miRNA-410可有效抑制视网膜新生血管的形成，进一步证明了miRNA-410在视网膜新生血管形成中所起的调控作用,同时为血管增生性视网膜病变的治疗提供了新的途径。
The retinal neovascular diseases, such as diabetic retinopathy, retinal vein occlusionand retinopathy of prematurity, can cause retinal neovascularization (RNV). Disease ofretinal ncovascular is one of the most serious diseases which can cause blindness in humanbeings all around the world. In developed country, the retinal neovascular disease is theleading cause of blindless. With the improved living standards, the retinal neovasculardisease has also become a threat to the visual health of our people. Retinalneovascularization can lead to vitreous hemorrhage，traction retinal detachment andneovascular glaucoma，even blindness, but the accurate patholgenesis is still unknown. Inrecent years, the roles of growth factors in the pathogenesis of RNV come to be theconsensus for most experts.
The imbalance of the neovascularization stimulating factor and inhibitory factor inlocal tissue play a vital role in producing new blood vessels. These stimulate blood vesselgrowth factors include vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF) and interlukin-8. The Inhibition of vascular endothelial growth factor includetransforming growth factor beta (TGF-β) and thrombospondin. VEGF is the main growthfactor which induced neovascularization in retina. We can reduce the formation of retinalneovascularization through inhibiting the expression of VEGF.
microRNAs (miRNAs) are endogenous, small, non-coding, regulatory RNAs,-22nucleotides (nts) in size. miRNAs target their downstream messenger RNAs by basepairing to their target sites with sequence complementarity, mainly in the3′untranslatedregion (UTR), and induce the breakdown of the targeted mRNAs and/or inhibition oftranslation from the mRNAs. There are studies shown that microRNAs play importantroles in cellular proliferation, differentiation, and cell death. microRNA involved in aseries of life processes through regulating gene expression. Approximately30%of theprotein-coding genes are estimated to be regulated by miRNAs. There are some progress inthe research of microRNA on oncology and cardiovascular. The scientists ofophthalmology are also attracted to the study of microRNA. Many miRNAs showedunique tissue-specific and developmental stage-specific expression patterns, suggestingpotential unique functions in the retina and other ocular tissues.
In this study,we aim to construct microRNA-410plasmid and investigate the effect ofmicroRNA-410on the inhibition of retinal neovascularization in mice of oxygen inducedretinopathy (OIR) and evaluate the efficiency of microRNA-410in retinal neovasculardisease．We use the condensed plasmid in two different ways. Firstly, intravitrealinjection was performed by inserting a needle into the vitreous of anesthetized OIR mice.Secondly, we make the condensed plasmid as the eye drops, drop it daily in the mouse eyesand observe the treatment effect. We found that microRNA-410inhibited the of retinalneovascularization in mice of OIR. The resultsshowed that miR-410reduced retinal NV inOIR model. The expression of VEGF was significantly reduced in the OIR model whichwas treated with miR-410, suggesting that miR-410may play a potential role in thepathogenesis in oxygen induced retinopathy.
Chapter one：Construction and Verification of miRNA-410Plasmid vector
Objective To construct miRNA-410Plasmid vector of mouse and verify theinhibition of vascular endothelial growth factor (VEGF) at the cellular level.
Methods miRNA-410recombinant plasmid was construct. Transfect themiRNA-410recombinant plasmids (cotransfection group) by the Lipofectamine2000method. Detect the expression of recombinant plasmid after transfection by Real-timePCR.Use RT-PCR to detect the expression change of VEGF in the Human Umbilical VeinEndothelial Cells（HUVEC）.
Results We constructed the miRNA-410recombinant plasmid(pLKO.1-miRNA-410) successfully. The expression of recombinant plasmids wereidentified by RT-PCR after24hours of transfection to the HUVEC. The level of VEGF ofthe pLKO.1-miRNA-410group is significantly downregulated than the pLKO.1-mock(p<0.05) in HUVEC. The level of VEGF of the pLKO.1-miRNA-410-antisense group issignificantly upregulated than the pLKO.1-mock group (p<0.05) in HUVEC.
Conclusions We constructed the miRNA-410recombinant plasmidsuccessfully and verify the inhibition of VEGF at the cellular level.
Chapter two： Establishment of a quantifying animal model ofretinal neovascularization and dynamic expression of VEGF
Objective To establish a quantifying model of retinal neovascularization suitablefor the study of pathogenesis and therapeutic intervention for the retinalneovascularization．
Methods One hundred7-day-old C57BL／6J mice were divided intooxygen-induced retinopathy group and control group．In oxygen-induced retinopathygroup，50mice were exposed to (75土2)％oxygen for5days and then to room air；incontrol group，50mice were raised in room air．Fourty mice of postnatal day13(P13),P15, P17, P19, P23were perfused and eyes were enucleated．The retinal preparationperfused with fluorescein-Dextran was examined to assess the oxygen-induced changes ofretinal vessels．The proliferated neovascular response was quantitated by counting thenuclei of endothelial cells of new vessels extending from the retina into the vitreous.Thedynamic expression of VEGF in the retinal was detected by real-time RT-PCR andImmunohistochemical detection.
Results The neovascularization in the eyes of oxygen-induced retinalpathy inmurine model was significantly increased compared with the normal group．There aremore construction and occlusion in retinal blood vessels and appears large tracts ofnon-perfused area.Similarly， histological analysis indicates that neovascular nucleiprotruding intoviteous cavity was increased compared to the normal group． Theneovascular nuclei protruding intoviteous cavity of oxygen-induced grouped is(1.39±1.12)，(16.67±3.51)，(42.31±4.69)，(39.23±4.5)，(0.93±0.85) in P13，P15，P17，P19，P23respectively，compared to normal group with value of (0.96±0.91)，(0.94±0.92),，(0.89±0.91)，(0.9±0.89)，(0.89±0.97) respectively.The expression of VEGFin oxygen-induced retinopathy group was dramatically increased than normal group.Thedifference was statistically significance (p<0.05).
Conclusions The reproducible and quantifiable mouse model of retinalneovascularization isuseful for the study of pathogenesis of retinal neovascularization andtherapeuticintervention．The expression of VEGFmRNA in mice with oxygen-inducedpreoliferative retinopathy is related to retinal anigogenesis.
Chapter three： Suppression of retinal neovascularization bymiRNA-410in Establishment of a quantifying animal model
Objective To evaluate the inhibitory effects of miRNA-410on retinalneovascularization in the mouse．
Methods Sixty7-day-old C57BL／6J mice were divided into oxygen-inducedretinopathy group and normal group．Ten Seven-day-old C57BL／6J mice were exposedto normal air randomly, fifty Seven-day-old C57BL／6J mice were exposed to (75±2)％oxygen for5days， then they were returned to the room air to induce retinalneovascularization．Ten of the oxygen-induced models were untreated.Ten of theoxygen-induced models were treated with eyedrops contains pLKO.1-miRNA-410.Ten ofthe oxygen-induced models were treated with eyedrops contains pLKO.1-mock.Ten ofthe oxygen-induced models were intrvitreally injected with pLKO.1-miRNA-410.Ten ofthe oxygen-induced models were intrvitreally injected with pLKO.1-mock. Retinalneovascularization was evaluated by angiography with injection of fluorescein dextran andthe proliferated neovascular response was quantitated by counting the nuclei of endothelialcells of new vessels extending from the retina into the vitreous.Moreover，RT-PCR wasused to detect the expression of VEGFmRNA in the retinas．
Results The neovascularization in the eyes of pLKO.1-miRNA-410intrvitrealinjection and pLKO.1-miRNA-410eyedrops group was significantly decreased comparedwith the oxygen-induced group．There are less construction and occlusion in retinal bloodvessels and appears smaller tracts of non-perfused area in this two groups. The expressionof VEGFmRNA in the two groups were dramatically decreased than that of theoxygen-induced group.The difference was statistically significance (p<0.05). Similarly， histological analysis indicates that neovascular nuclei protruding intoviteous cavity wasdecreased compared to the oxygen-induced group．
The neovascularization in the eyes of pLKO.1-miRNA-mock intrvitreal injection andpLKO.1-miRNA-mock eyedrops group was similar to the oxygen-induced group．Thereare many occlusions in retinal blood vessels and appears lage tracts of non-perfused area inthis two groups. The expression of VEGFmRNA in the two groups were dramaticallyincreased than that of the normal group.The difference was statistically significance(p<0.05). Similarly,histological analysis indicates that neovascular nuclei protrudingintoviteous cavity was increased compared to the normal group．The difference wasstatistically significance (p<0.05).
Conclusions Retinal neovascularization can be inhibited by intravitreal injectionof pLKO.1-miRNA-410,which suggests that intravitreal injection of pLKO.1-miRNA-410may have potential therapeutic benefits in ischemic-induced retinal diseases. miRNA-410can reduce the expression of VEGF in the retinas of mice．It may provide a powerful andnovel therapeutic tool for ischemic-induced retinal diseases．
引文
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