高氧诱导新生鼠视网膜VEGF及TNF-α的表达与地塞米松的干预研究

发布时间：2018-03-01 16:18

本文关键词： 早产儿视网膜病新生血管地塞米松血管内皮生长因子肿瘤坏死因子-α　出处：《广州医学院》2012年硕士论文　论文类型：学位论文

【摘要】：作为世界范围内儿童致盲的重要原因之一，早产儿视网膜病(ROP)一直是大家关注的焦点，多发生于早产儿和低体重儿中，是一种视网膜血管增生性病变。ROP的发病原因与早产儿视网膜本身血管的发育不完全有关，当视网膜因多种原因形成新生血管以及伴有纤维化时，即出现增殖性视网膜病变，严重者可发生牵引性视网膜脱离，所以研究新生血管的发生机制近年来成为学界重点关注的方向，预防及治疗早产儿视网膜病的研究切入点是如何抑制视网膜新生血管的形成。早产儿视网膜病的严重并发症是因视网膜血管的异常增生引起，而视网膜血管内皮细胞的增殖受视网膜血管形成刺激因子和血管形成抑制因子的共同调控，达到一种平衡状态，该状态一旦被打破，即可启动新生血管生长。国外研究发现新生血管的生长多是由于上调血管促进因子或下调血管抑制因子所致，而这些因子主要包括有血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)、色素上皮衍生因子（PEDF）等。其中VEGF占据着至关重要的地位，无论在正常或者异常状态下对于血管的形成均具有重要作用，在视网膜血管正常发育以及成熟过程中VEGF起着促进作用，在ROP病变中视网膜血管质和量的变化可以推测是由于VEGF在视网膜表达水平的变化所引起。除了VEGF外，TNF—α作为另外一种重要的血管诱导因子，在炎症、细胞凋亡和血管形成过程中也发挥着重要作用。因此，视网膜新生血管抑制因子疗法已成为具有颇大研究前景的ROP诊治方法之一。近年来国内外均有报道地塞米松用于一些视网膜疾病如急性前葡萄膜炎、视网膜冲击伤、增殖性糖尿病视网膜病及角膜碱烧伤等的治疗。目前临床上广泛产前应用糖皮质激素地塞米松促进胎肺成熟，而地塞米松对于早产儿视网膜病变的发生发展有何影响及其作用机制国内外尚缺乏令人信服的实验依据。目的模拟早产儿视网膜病(retinopathy of prematurity，ROP)的病理特点，建立高氧诱导视网膜病的新生鼠模型，为研究ROP的发病机制及治疗方法提供实验基础。观察地塞米松对高氧诱导小鼠视网膜新生血管的形成有何影响，探讨地塞米松对新生血管形成是否存在抑制作用及其可能的作用机制，并研究不同剂量下地塞米松对ROP的影响，为地塞米松可能影响早产儿视网膜病的发生发展提供实验依据。方法一、高氧诱导新生鼠视网膜血管增生模型的建立：选择7曰龄C57BL/6J新生小鼠58只，分为单纯高氧诱导组和对照组，将29只单纯高氧诱导组小鼠置于特制密闭容器（内环境控制在氧浓度为75％）中饲养5天，随后放回至正常空气环境下饲养，17日龄时处死；另29只对照组小鼠持续在正常空气环境中生长到17日龄。两组新生鼠在鼠龄分别为P12(生后第12天)、P14时各处死8只，其余的在P17时处死，处死前称重。两组视网膜铺片ADP酶染色后，比较视网膜血管形态、走向及分布上的差异；两组视网膜切片HE染色后，观察并计数突破视网膜内界膜的血管内皮细胞核数目，从而定量反映视网膜血管的增生情况。二、地塞米松影响鼠视网膜新生血管生成和VEGF、TNF—α表达的研究：建立高氧大剂量地塞米松组、高氧小剂量地塞米松组、高氧对照组及评价组，每组小鼠各15只。在P12、P14和P17时称重，并于17日龄时处死。各组视网膜铺片经ADP酶染色后，观察视网膜血管的改变；视网膜病理切片HE染色后，计数突破视网膜内界膜的新生血管内皮细胞核数目；VEGF蛋白及TNF—α蛋白在各组小鼠视网膜中表达的强度及分布。结果一、高氧诱导新生鼠视网膜血管增生模型的建立： 1.对照组在P17时视网膜血管的发育大致趋向成熟，血管网结构清晰，由视盘发出的大血管呈放射状，向四周均匀分布散开，管径逐渐由粗变细直至视网膜周边部，未见血管闭塞。单纯高氧诱导组在P17时由视盘发出的视网膜血管出现明显扩张、迂曲，在中周部可见较多新生血管，这些血管的密度增高，血管网的结构和分布均比较紊乱，大片无灌注区存在于后极部视网膜。 2.对照组未发现或仅在非常少数的切片中发现有突破视网膜内界膜血管内皮细胞核，而单纯高氧诱导组可见较多突破视网膜内界膜的血管内皮细胞核，有的单独出现，有的成簇状，有的形成新生血管管腔；高氧诱导组内突破视网膜内界膜的血管内皮细胞核数P14、P17分别与P12比较均有统计学意义（P0.01），而两组比较在P12时无差异，在P14、P17时差异有统计学意义（P0.01）。 3.单纯高氧诱导组在P17时新生鼠视网膜切片中新生血管内皮细胞核数左右眼相比较无统计学意义。二、地塞米松影响鼠视网膜新生血管生成和VEGF、TNF—α表达的研究： 1. ADP酶染色：大、小剂量地塞米松组视网膜血管形态与高氧对照组相比明显正常，更接近正常对照组的血管形态，无灌注区明显缩小，新生血管不明显，说明两组不同剂量的地塞米松对新生血管的生长均有明显抑制作用。评价组的视网膜血管与对照组相比，差异无统计学意义，说明地塞米松对正常血管的生长抑制作用不明显。 2.HE染色：两组不同剂量地塞米松组眼组织切片中仅见少量突破视网膜内界膜的血管内皮细胞核，高氧对照组所有切片均可见突破内界膜的内皮细胞核，与高氧诱导组相类似。各组镜下观察突破视网膜内界膜的血管内皮细胞核数，大、小剂量地塞米松组分别和高氧对照组比较，差异均有统计学意义。但地塞米松不同剂量组之间比较，P0.05，两组差异无统计学意义。 3.免疫组化分析：在P17时，对照组VEGF蛋白主要在视网膜神经纤维层、神经节细胞层、色素上皮层（RPE层）呈弱阳性表达；TNF—α蛋白也呈弱阳性，，阳性信号主要分布于内核层、视网膜神经节细胞层。单纯高氧诱导组和高氧对照组VEGF蛋白及TNF—α蛋白均呈强阳性表达，其中VEGF蛋白在视网膜各层均有表达，而TNF—α蛋白则主要分布于新生血管内皮细胞、内界膜层、神经节细胞层和内核层。大、小剂量地塞米松组中VEGF及TNF—α的表达较单纯高氧诱导组明显减弱。 4.大、小剂量地塞米松组与对照组相比较，在P7时体重无明显差异，而在P12、P17时体重差异均有统计学意义。结论 1.成功建立了ROP的小鼠模型，为进一步研究药物治疗早产儿视网膜病提供了比较合适的动物模型。 2.在高氧诱导视网膜病变的小鼠模型中，VEGF蛋白、TNF—α蛋白在视网膜上的表达增强，作为氧诱导视网膜病变的重要血管影响因子，它们的表达与视网膜血管新生有着密切的关系。 3.地塞米松对视网膜新生血管的形成可能有抑制作用，影响高氧诱导新生鼠视网膜新生血管生成过程，对视网膜起着一定的保护作用。 4.地塞米松影响视网膜新生血管形成的作用机制可能与其抑制了视网膜VEGF蛋白及TNF—α蛋白表达有关，这为进一步研究ROP的预防和治疗开辟了新方法。
[Abstract]:As one of the important causes of blindness in children all over the world, retinopathy of prematurity (ROP) has been the focus of attention, mainly occurs in premature and low birth weight infants, is a cause of disease and retinopathy of prematurity of vascular proliferative retinopathy.ROP itself vascular development related to incomplete, when retinal neovascularization and fibrosis form for various reasons, namely proliferative retinopathy, serious person can produce traction retinal detachment, so the study of mechanism of angiogenesis in recent years has become the academic focus on the direction of research, prevention and treatment of retinopathy of prematurity is the cut in point form how to inhibit retinal neovascularization.
Serious complications of retinopathy of prematurity is caused by abnormal proliferation of retinal vessels and retinal vascular endothelial cell proliferation by retinal angiogenesis stimulating factor and angiogenesis inhibiting factor co regulation, to reach a balance state, the state once it is broken, you can start the growth of new blood vessels. The foreign study found that the growth of new blood vessels is due to the upregulation of vascular promoting factor or downregulation of angiogenesis inhibitory factor caused by these factors including vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF- alpha), pigment epithelium derived factor (PEDF). The VEGF plays a crucial role, either in normal or abnormal condition for the formation of blood vessels have an important role, VEGF plays a role in promoting the normal development of the retinal vessels and the maturation of the lesions in ROP in vascular net film quality and quantity The change may be speculated to be due to changes in the expression level of VEGF in the retina caused. In addition to VEGF, TNF - alpha as another important factor in angiogenesis, inflammation, apoptosis and angiogenesis also plays an important role. Therefore, retinal angiogenesis inhibitors therapy has become one of the methods of diagnosis and treatment of ROP with considerable research prospect.
In recent years at home and abroad have reported dexamethasone for some retinal diseases such as acute anterior uveitis, retinal blast injury, the treatment of proliferative diabetic retinopathy and corneal alkali burn. The widely clinical application of prenatal dexamethasone promoting fetal lung maturation, and dexamethasone for the occurrence and development of retinopathy of prematurity and the impact of domestic role the mechanism is still a lack of convincingevidence.
objective
Simulation of retinopathy of prematurity (retinopathy of, prematurity, ROP) of the pathological characteristics, establishment of neonatal rat model of oxygen induced retinopathy, provide the experimental basis for studying the pathogenesis and treatment of ROP. What is the effect of dexamethasone on the formation of high oxygen induced retinal neovascularization in mice, to investigate whether dexamethasone inhibitory effect and mechanism of existence the possible formation of new blood vessels, and to study the effects of different doses of dexamethasone on ROP, provide experimental basis for the effect of the occurrence and development of retinopathy of prematurity for dexamethasone.
Method
First, establish a neonatal rat model of oxygen induced retinal neovascularization: 7 day old neonatal C57BL/6J 58 mice were divided into pure oxygen induced group and control group, 29 pure oxygen induced mice were placed in a special airtight container (environmental control in 75% oxygen concentration) in the feeding for 5 days. Then back to normal air feeding, were sacrificed at the age of 17 days; the other 29 mice in control group continued growth to 17 days of age in the normal environment. The two groups of neonatal rats in rat age were P12 (postnatal day twelfth), P14 8 rats of each group were sacrificed, the rest were killed at P17 before death, weighing two group. Retinal flatmount ADP staining after comparing the morphology of retinal vessels, and to the difference of distribution of the two groups; retinal sections after HE staining to observe and count the number of vascular endothelial cells in retinal, and quantitatively reflect the proliferation of retinal vessels.
Two, the effect of dexamethasone in retinal angiogenesis and VEGF expression of TNF alpha: a high oxygen high dose dexamethasone group, high oxygen and low dose dexamethasone group, hyperoxia group and evaluation group, each group of 15 mice each. Only in P12, weighing P14 and P17, and were sacrificed at 17 days of age each. The retinas by ADP staining, observe the changes of retinal vessels; retinal pathological HE staining, neovascularization of endothelial cell count the number of retinal VEGF protein; and the expression of TNF protein in mice retina in intensity and distribution.
Result
1. The establishment of hyperoxia induced retinal vascular proliferation model in neonatal rats.
1. P17 in the control group when the development of retinal vessels generally mature vascular network, clear structure, large vessels from the optic disc from the radial, around to evenly spread, the diameter gradually tapering to peripheral retinal vascular occlusion, no. Pure oxygen induced retinal blood vessels in P17 group by a disc significant expansion in the periphery, tortuous, more visible neovascularization, the vascular density, structure and distribution of vascular network was disorder, avascular area in the posterior retina.
2. the control group was not found or only in very few sections were found in retinal vascular endothelial cells, while the group showed more retinal oxygen induced simple vascular endothelial cells, some alone, some tufted, some form of blood vessels; retinal vascular endothelial the number of nuclei of P14 induced by hyperoxia group, P17 and P12 were statistically significant (P0.01), and the comparison between the two groups in P12, no differences in P14, P17 had significant difference (P0.01).
3. there was no significant difference in the number of the nuclei of the neovascular endothelial cells in the retinal slices of the newborn rats at P17.
Two, dexamethasone affects the formation of retinal neovascularization and the expression of VEGF, TNF - alpha in the retina of rats.
1. ADP staining: large, small doses of dexamethasone group, retinal vascular morphology and high oxygen control group significantly than normal vascular morphology, close to the normal control group, no significantly reduced perfusion and neovascularization is not obvious, that the growth of both the two groups of different doses of dexamethasone on angiogenesis is inhibited. Compared to the evaluation group retinal blood vessels and the control group, the difference was not statistically significant, indicating the growth inhibitory effect of dexamethasone on normal blood vessels is not obvious.
2.HE staining: only two groups of different doses of dexamethasone eye tissue sections in small retinal vascular endothelial cells in hyperoxia group, all sections were visible through the internal limiting membrane of endothelial cells, and high oxygen induced group of similar groups. Under microscope through endothelial cell nuclei, retinal inner limiting membrane the large and small dose dexamethasone group and hyperoxia compared to the control group, there were statistically significant differences between the different doses of dexamethasone. But compared to P0.05, there was no significant difference between the two groups.
3. immunohistochemical analysis: in P17, the control group of VEGF protein mainly in the retinal nerve fiber layer, ganglion cell layer and pigment epithelium layer (RPE layer) showed weak positive expression; TNF protein was weakly positive, the positive signals were mainly distributed in the inner nuclear layer, retinal ganglion cell layer with high oxygen induced group. And the hyperoxia group VEGF protein and TNF protein showed strong positive expression, the expression of VEGF protein in retinal layers were TNF protein mainly distributed in vascular endothelial cells, inner layer, ganglion cell layer and kernel layer. Large group was significantly decreased induced by low-dose dexamethasone group VEGF and TNF expression compared with hyperoxia.
The 4. major, small dose of dexamethasone group compared with the control group, there was no significant difference in weight at P7, but the weight difference was statistically significant at P12 and P17.
conclusion
1. the mouse model of ROP was successfully established, which provides a more suitable animal model for the further study of the treatment of retinopathy in preterm infants.
2., in the hyperoxia induced retinopathy model, the expression of VEGF protein and TNF - alpha protein on the retina is enhanced. As an important vascular factor of oxygen induced retinopathy, their expression is closely related to retinal angiogenesis.
3., dexamethasone may inhibit the formation of retinal neovascularization, influence the process of retinal angiogenesis induced by hyperoxia, and play a protective role in retina.
4., the mechanism of the effect of dexamethasone on retinal neovascularization may be related to the inhibition of the expression of retinal VEGF protein and TNF - alpha protein, which opens a new way for further research on the prevention and treatment of ROP.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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