早产对大鼠视网膜发育及氧诱导视网膜病变的影响

发布时间：2018-05-16 20:29

本文选题：早产儿视网膜病变 + 氧诱导视网膜病变　；参考：《第四军医大学》2013年博士论文

【摘要】：研究背景早产是一种常见的临床现象，导致新生儿较高的死亡率和发病率。临床证据表明，早产儿发生视觉损害和眼部疾病的风险明显高于足月儿。早产可影响视网膜血管系统的结构和功能发育，从而使得早产儿罹患血管性疾病的阈值降低，临床上最为常见的表现是早产儿视网膜病变（ROP）。ROP是一种复杂的视网膜新生血管性疾病，因早产儿的血管发育异常所致。视网膜血管化越不成熟，发生严重ROP的风险就越大。小胎龄和低出生体重是公认的发生ROP的最重要的危险因素。但是，作为一种多因素致病性疾病，ROP的病因和病理机制目前尚未阐明。大鼠氧诱导视网膜病变（OIR）模型广泛用于ROP的实验研究中，其应用基础为新生大鼠发育不成熟的视网膜血管系统与人类早产儿极其相似。然而，OIR采用的是足日龄生产的正常新生大鼠，它们出生时和出生后的病理生理状态与早产儿有所不同。在新生大鼠中模拟早产儿出生后生长受限的研究发现，出生后的生长发育情况决定了幼鼠正常视网膜血管化的速度和OIR中异常血管化的程度。理论上，早产大鼠可在一定程度上模拟人类早产儿出生时的不成熟以及出生后的发育不良经历，能为研究“早产”这一因素在ROP中的作用提供一个良好的平台。虽然早产大鼠模型已被广泛用于研究人类发育相关性疾病，但目前有关早产大鼠的视网膜发育特点以及早产对大鼠OIR的影响尚无报道。目的 1.观察早产大鼠的神经视网膜、视网膜浅层毛细血管的结构以及视网膜功能的发育特点； 2.观察早产大鼠在氧诱导条件下的视网膜血管病变情况及视网膜中血管内皮生长因子（VEGF）和胰岛素样生长因子1（IGF-1）的表达水平。方法 1.于Sprague Dawley（SD）大鼠孕19天时行剖宫产，建立早产大鼠模型，观察空气中饲养的早产幼鼠的一般情况及体重变化。同时设正常足日龄生产的大鼠作为对照。 2.分别于幼鼠出生后4、7、10、14天行视网膜切片、HE染色及荧光标记的isolectin B4视网膜血管染色、铺片，观察幼鼠视网膜各层细胞和浅层血管形态，测量神经视网膜各层厚度和未血管化区面积；分别于幼鼠出生后14、21、28、35天行视网膜电图（ERG）检测，记录各波的波幅和潜伏期。 3.将新生大鼠在出生后6h内置于80%/21%浓度、24h交替的动物氧箱中，维持2周，于出生后15天返回空气中饲养，建立大鼠OIR模型；分别于出生后14、18、22天行荧光标记的isolectin B4视网膜血管染色、铺片，观察视网膜血管形态，测量视网膜无血管区及新生血管区面积；采用反转录多聚酶链式反应（RT-PCR）检测视网膜中VEGF、IGF-1mRNA的表达水平；于出生后18天行视网膜切片、HE染色，计数突破视网膜内界膜的血管内皮细胞数量。结果 1.剖宫产手术的成功率以及早产幼鼠的存活率较高。出生后3周内，早产幼鼠的体重低于足日龄幼鼠（P0.001）。 2.早产幼鼠的视网膜各层细胞和浅层血管形态及发育趋势与对照组相似；出生后早期，早产幼鼠的视网膜厚度大于对照组（P0.05），2周时趋于一致；2周内早产幼鼠的视网膜无血管区明显大于对照组（P0.001），2周时两组的视网膜血管化均已完成；出生2周后，早产鼠的ERG波幅和潜伏期与对照组相似。 3.波动氧干预后，早产幼鼠的视网膜一级血管迂曲程度与对照组相似；早产幼鼠14和18天的视网膜无血管区及18天的视网膜新生血管区面积大于对照组（P0.001），早产幼鼠22天时残留的视网膜血管病变较对照组更多（P0.01）。早产幼鼠14、18、22天的视网膜中VEGF mRNA的表达高于对照组，，IGF-1mRNA的表达无明显差异。结论剖宫产诱导的早产对幼鼠的体重增长及神经视网膜、视网膜血管发育产生负性影响。早产幼鼠不成熟的视网膜血管系统更易遭受氧气的损伤，导致更严重而持久的视网膜病变。这些研究结果将为进一步在动物模型中研究各种危险因素在ROP发生过程中的“多重作用”提供基础数据。
[Abstract]:Background of the study

Premature delivery is a common clinical phenomenon , leading to higher mortality and morbidity in neonates . The clinical evidence suggests that preterm infants have a higher risk of visual impairment and eye disease than in term infants . Early birth can affect the structural and functional development of retinal vasculature , which is the most important risk factor for preterm infants . However , as a multi - factor pathogenicity disease , the etiology and pathological mechanism of ROP has not yet been elucidated .

In the experimental study of rat ' s oxygen - induced retinopathy ( OIR ) model widely used in ROP , the application of OIR is very similar to that of preterm infants .

Purpose

1 . To observe the structure of retinal and retinal shallow capillary vessel and the development of retinal function in preterm rats .

2 . The expression level of vascular endothelial growth factor ( VEGF ) and insulin - like growth factor 1 ( IGF - 1 ) in the retina of preterm rats under oxygen - induced conditions were observed .

method

1 . In Sprague Dawley ( SD ) rats , a model of preterm rats was established to observe the general condition and body weight change of preterm young rats fed in air .

2 . At 4 , 7 , 10 and 14 days after birth , retinal slices , HE staining and fluorescent labeled isolectin B4 were stained , and the thickness of retinal layers and the area of non - vascularization area were measured .
Electroretinogram ( ERG ) was detected at 14 , 21 , 28 and 35 days after birth respectively , and the amplitude and latency of each wave were recorded .

3 . The neonatal rats were placed in 80 % / 21 % concentration at 6 h after birth , in an alternate animal oxygen tank for 2 weeks , returned to air for 15 days after birth , and the rat OIR model was established ;
At 14 , 18 and 22 days after birth , the retinal vessels were stained with the fluorescent labeled isolectin B4 , and the retinal vessel morphology was observed and the area of retinal blood vessel and neovascularization was measured .
The expression level of VEGF and IGF - 1 mRNA in the retina was measured by reverse transcription polymerase chain reaction ( RT - PCR ) .
The retinal section and HE staining were performed on 18 days after birth , and the number of vascular endothelial cells was counted .

Results

1 . The success rate of the caesarean operation and the survival rate of the preterm young rats were higher . In the 3 weeks after birth , the weight of the premature infant was lower than that of the young mouse ( P0.001 ) .

2 . The morphology and developmental tendency of retinal layer cells and superficial layers of early mouse were similar to those of the control group .
The retinal thickness of early and early postnatal mice was greater than that of control group ( P0.05 ) , and tended to be consistent in 2 weeks .
There was no significant difference between the two groups ( P 0.001 ) , and the retinal vascularization in two groups was completed in 2 weeks .
After 2 weeks of birth , the ERG amplitude and latency of the preterm mice were similar to those of the control group .

3 . After the intervention of the fluctuating oxygen , the degree of the retinal primary vessel was similar to that of the control group .
Compared with the control group ( P0.01 ) , the expression of VEGF mRNA was higher in the retina without vascular and 18 days earlier than that in the control group ( P0.01 ) . The expression of VEGF mRNA in the retina of early - birth young rats was higher than that in the control group .

Conclusion

The results of these studies will provide basic data for further study of " multiple effects " of various risk factors in the process of ROP .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R722.6

【参考文献】