miRNA-410对小鼠视网膜新生血管抑制作用的研究

发布时间：2018-05-10 18:06

本文选题：小分子RNA + 基因表达　；参考：《第二军医大学》2012年博士论文

【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(p0.05),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(p0.05)。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(p0.05)。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(p0.01)。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(p0.05)；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(p0.05)，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(p0.05)；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(p0.05)；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（p0.05）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（p0.05）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。
[Abstract]:Retinal neovascularization, such as diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity, can all lead to neovascularization of the retina. At present, retinal neovascularization is one of the most serious blindness diseases in the world, and is the primary cause of blindness in the country of hair. With the improvement of the living standard of the people in our country, the new vascular diseases in the fundus have become an important cause of the people's vision health. The formation of retinal neovascularization can cause vitreous hemorrhage, traction retinal detachment and neovascular glaucoma, its pathogenesis is not completely clear. However, the growth factor has been in the optic network in recent years. A consensus has been made on the role of neovascularization in the membrane. The maladjustment of the dynamic balance of neovascularization and inhibitory factors in local tissue is the key to the production of neovascularization. The factors that have been found to stimulate vascular growth include vascular endothelial growth factor (vascular endothelial growthfactor, VEGF), fibroblast growth factor (fibrob Last growth factor, FGF), interleukin 8 (interlukin-8), and so on. The factors that have been found to inhibit vascular growth are transforming growth factors (transforming growthfactor beta, TGF- beta) and thromboxane (thrombospondin), etc..VEGF is the most important growth factor in retinal neovascularization. Although the mechanism of retinal neovascularization has not been completed yet Although many regulatory factors are still unclear, many studies have shown that inhibiting the expression of VEGF can reduce the formation of retinal neovascularization.
MicroRNA (miRNA) is an endogenous, non coded single strand small molecule RNA, which is paired with the 3 'non translation region of the target gene mRNA to play a role in the degradation of mRNA or to inhibit the translation of mRNA, thus regulating the post transcriptional expression of the gene. A large about 30% of the protein encoding genes are.MiRNA regulated by.MiRNA. The important role of.MicroRNA in cell proliferation, differentiation and apoptosis, as well as all the biological processes reported, has made some progress in the research of tumor, cardiovascular and other aspects. Its application in the ophthalmology has also aroused the interest of scholars. Studies have found that the expression of microRNA in the retina and other tissues of the eye has been found. Tissue-specific and developmental stage specificity suggests that it has potential tissue and cell function specificity in the retina and other tissues of the eye.
In this experiment, the mouse microRNA-410 plasmid vector was constructed and the mouse retinal neovascularization model induced by hyperoxia was used as the experimental object. The condensed plasmids were constructed in two different ways: 1. vitreous cavity was injected into the eye of the mice by injection of plasmids, and the concentrated plasmid was made into eye drops after 5 days. The effect of local eye drops for 5 days was observed. The results showed that microRNA-410 could inhibit the proliferation of vascular endothelial cells by down regulating the expression of VEGF and reduce the number of retinal neovascularization, thus hindering the formation of neovascularization.
Part 1 construction and verification of miRNA-410 plasmid vector
Objective to construct miRNA-410 plasmid vector and to verify its inhibitory effect on VEGF at cellular level.
Methods the recombinant expression plasmid (pLKO.1-miRNA-410) of miRNA-410 was constructed, and the expression plasmid and empty plasmid were transferred into human umbilical vein endothelial cells (HUVEC) by liposome method. The expression of the recombinant plasmid was detected by RT-PCR method, and the changes of VEGF expression were detected by real-time quantitative PCR and Western blot.
Results the recombinant plasmid of miRNA-410 was successfully constructed. The expression of VEGF in the transfected HUVEC was significantly lower than that in the transfected pLKO.1-mock (P0.05). The expression of VEGF in pLKO.1-miRNA-410-antisense's HUVEC was obviously up regulated than that in the transfected pLKO.1-mock (P0.05).
Conclusion miRNA-410 recombinant plasmid was successfully constructed, and its inhibitory effect on VEGF was verified at cell level.
The second part is the establishment of quantitative oxygen induced retinal neovascularization in mice and the dynamic expression of VEGFmRNA.
Objective to establish an animal model for quantitative study of retinal neovascularization, and to provide an experimental basis for the next study of the mechanism and treatment of retinal neovascularization.
Methods the healthy 7 days old (P7) C57BL/6J mice were selected and 16 litters and 100 rats were randomly divided into hyperoxia group (group OIR) and control group (group C). 50 7 day old C57BL / 6J mice (P7) were placed in the oxygen concentration environment with a concentration of (75 + 2)% in the concentration of (75 + 2)%), and returned to the normal air after 5 days as the oxygen induced model group; the control group: the control group: 50 newborn mice of the same age were placed in the normal air environment as the normal control group. The two groups were killed in P13, P15, P17, P19, P23, respectively. The eyeball was removed and the retinal neovascularization was observed by the FITC-Dextran fluorescein retina sheet. The tissue sections were observed and counted in the sagittal 5mm retina slices. The number of endothelial cells in the inner limiting membrane reflects the proliferation of retinal vessels, and the dynamic expression of VEGFmRNA in the retina is detected by real-time RT-PCR.
Results the retinal vasculature of fluorescein perfusion method showed that the retinal vessels in the normal group showed a uniform network, no neovascularization and no perfusion area. After 1 days (P13) of the high oxygen induction group, the retina began to appear in the retina. After 3 days (P15) in the air, the neovascularization of the retina was increased obviously. After 5 days (P17) in the air, the formation of retinal neovascularization reached its peak. After 7 days (P19) in the air, the neovascularization of the retina decreased gradually. After 12 days (P23) in the air, the neovascularization of the retina subsided. The retinal section was broken through the number of vascular endothelial cells in the inner retina membrane, and the model group induced by P13.P15.P17.P19.P23 oxygen was respectively (1.39 + 1.12), (16.67 + 3.51), (42.31 + 4.69), (39.23 + 4.5), (0.93 + 0.85), normal control group (0.96 + 0.91), (0.94 + 4.69), (4.69), compared with the difference (P0.05). The expression of the retinal tissue at each time point in the hyperoxic induced model group The level of the control group was significantly higher than that of the control group at the same time point, and the difference between the two groups was significant (P0.01).
Conclusion the model rate is high, the reproducibility is good, and the quantitative study can be carried out. It is a suitable model for the study of the mechanism of retinal neovascularization and the study of drug therapy in mice retina. The result of.RT-PCR shows that VEGFmRNA surface Daming in retinal tissue of mice with oxygen induced proliferative retinopathy is up to be up-regulated. The formation of blood vessels should be corresponding.
The third part of miRNA-410 inhibits retinal neovascularization in mice.
Objective To observe the inhibitory effect of miRNA-410 on retinal neovascularization, and further clarify the regulatory role of miRNA-410 in inhibiting retinal neovascularization.
Methods the healthy 7 days old (P7) C57BL/6J mice were selected and male and female were not limited. A total of 10 nests and 60 C57BL / 6J mice were selected. 10 C57BL / 6J mice fed in normal oxygen environment were randomly selected as normal control group. The remaining 50 mice aged 7 days of C57BL / 6J mice were placed in the concentration of (75 + 2)% hyperoxic environment for 5 days, and returned to normal oxygen environment in P12. Of the 50 mice treated with hyperoxia, 10 were taken randomly, without any treatment, as a hyperoxic induction model group. The remaining 40 mice removed from the hyperoxic environment were randomly selected from 10 mice in the oxygen chamber and at the local point of pLKO.1-miRNA-410 eye daily in the right eye, and 2/ days as the pLKO.1-miRNA-410 eye drops. 10 mice were randomly selected. After the oxygen chamber, pLKO.1-mock eye drops in the right eye, 2/ day, as pLKO.1-mock eye drops,.10 mice were injected with 0.4 mu lpLKO.1-miRNA-410 in the right eye glass cavity of the right eye on the day after the oxygen capsule, as the intraocular injection pLKO.1-miRNA-410 group, and the 10 mice were injected with 0.4 Mu L pLKO.1-mock in the right eye glass cavity on the same day after the oxygen chamber. The left eye of the 5 groups of mice were not treated with exogenous treatment. All the mice in the above 5 groups were not treated with exogenous treatment. All the mice were harvested in the state of anesthesia at P17. The number of vascular endothelial nuclei in the inner boundary membrane of the retina was observed by histological section, and the changes of the retinal vascular morphology of the retina were detected by FITC-Dextran fluorescein angiography; RT-PCR was used to detect the retina. The expression level of VEGFmRNA in the tissue.
Results the results of histological section showed that the number of vascular endothelial nuclei in the hyperoxia induced group was significantly different from that of the normal group (P0.05), and the ratio of the local use of pLKO.1-miRNA-410 eye drug group and the intravitreal injection pLKO.1-miRNA-410 group with the hyperoxia induction group was significant (P0.05), and the difference was worse than that of the normal group. There was no significant difference (P > 0.05); there was a significant difference between the local use of pLKO.1-miRNA-410 eye drops and the local use of pLKO.1-miRNA-mock eye drops (P0.05), and there was a significant difference between the pLKO.1-miRNA-410 group and the pLKO.1-miRNA-mock group injected with the vitreous cavity (P0.05), and the local use of pLKO.1-miRNA-410 eye medicine group (P0.05). There was no significant difference between pLKO.1-miRNA-410 group and intravitreal injection group (P > 0.05).
The results of retina spread showed that the hyperoxia induced model group was injected into the pLKO.1-miRNA-410 group by injection of the vitreous cavity, and the new vascular plexus of the pLKO.1-mock group was reduced and the leakage was lightened. The high oxygen induction group in the pLKO.1-miRNA-410 eye medicine group, the pLKO.1-miRNA-410 group injected with the glass cavity, the intravitreal injection in the vitreous cavity. The neovascularization group of pLKO.1-mock group and local pLKO.1-mock eye drops group decreased significantly and fluorescence leakage was lightest.
VEGFmRNA level detection in retinal tissue: 1. the expression of VEGFmRNA in group pLKO.1-miRNA-410 injected with vitreous cavity was lower than that of intravitreal injection of pLKO.1-mock. The difference was significant (P0.05) 2. local use of pLKO.1-miRNA-410 eye medicine group and local use of pLKO.1-mock ophthalmic water group, the expression of VEGFmRNA decreased. The difference was significant (P0.05) 3. Compared with intravitreal injection of pLKO.1-miRNA-410, the expression of VEGFmRNA in local pLKO.1-miRNA-410 eye drops was not significantly different (P > 0.05).
Conclusion miRNA-410 can effectively inhibit the formation of retinal neovascularization, and further demonstrate the regulatory role of miRNA-410 in the formation of retinal neovascularization, and provide a new way for the treatment of vascular proliferative retinopathy.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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