宫内炎症致脑白质损伤新生大鼠的脑组织EPO及EPOR mRNA的表达及rhEPO的治疗作用研究

发布时间：2018-06-13 07:42

本文选题：促红细胞生成素 + 脑白质　；参考：《青岛大学》2015年博士论文

【摘要】：目的孕鼠腹腔注射脂多糖(lipopolysaccharide,LPS)制作宫内炎症致新生鼠脑白质病变动物模型。对脑白质病变新生鼠腹腔注射重组促红细胞生成素(recombinant human erythropoietin,rh EPO)rh EPO,通过内源性EPO/EPOR m RNA及CD68、GFAP、MBP的检测和神经行为学评分,探讨rh EPO对新生大鼠脑白质病变近期和远期的神经保护作用,为临床应用rh EPO治疗早产儿脑白质病变提供依据。方法30只Wistar孕鼠(孕龄15天)随机数字法分为实验组和对照组。实验组腹腔注射LPS 300μg/kg制作早产儿脑白质病变动物模型,对照组Wistar大鼠腹腔注射生理盐水(Normal saline,NS)1m L/kg作为对照。新生大鼠出生后,实验组与对照组各选30只新生鼠4%多聚甲醛灌注取脑,左侧脑组织进行HE染色,明确脑白质损伤情况;右侧脑组织用于实时荧光定量RT-PCR法检测胼胝体促红细胞生成素(erythropoietin,EPO)及EPOR m RNA水平。实验组随机数字法取60只新生鼠随机分为LPS+rh EPO组(n=30)和LPS+NS组(n=30),分别于出生后即刻一次性腹腔注射rh EPO(5000IU/kg)和生理盐水(1m L/kg)。对照组随机数字法取60只新生鼠随机分为NS+rh EPO组(n=30)和NS+NS组(n=30),分别于出生后即刻腹腔注射rh EPO(5000IU/kg)和生理盐水(1m L/kg)。各处理组分别于用药后后第3天、第7天随机取10只新生鼠,进行脑组织HE染色,免疫荧光法进行CD68、GFAP及MBP的检测。出生后2周各处理组随机选取10只大鼠进行神经行为学评分。结果1.孕15天Wistar大鼠腹腔注射LPS后所产新生鼠HE染色脑室周围脑白质染色淡染,结构稀疏,呈网状改变;对照组脑白质组织结构正常。2.腹腔注射LPS大鼠所产新生鼠胼胝体EPO m RNA及EPOR m RNA的表达明显高于腹腔注射NS组(P0.01)。3.生后3天HE染色显示LPS+rh EPO组新生大鼠脑室周围脑白质染色淡染,结构稀疏,呈网状改变;LPS+NS组新生大鼠脑白质结构稀疏,较LPS+rh EPO组加重。生后7天,HE染色显示LPS+rh EPO组脑室周围白质结构稀疏,染色较清晰;LPS+NS组脑白质结构稀疏,呈网状改变,染色不清晰。NS+rh EPO组及NS+NS组生后3天及7天脑白质组织结构正常,染色清晰。4.生后3天,LPS+rh EPO组、NS+rh EPO组、NS+NS组CD68表达均较LPS+NS组显著下降(F=7.456,P0.01),生后7d,LPS+rh EPO组、NS+rh EPO组、NS+NS组GFAP表达明显低于LPS+NS组(F=5.121,P0.01),生后3天(F=2.628)、7天(F=1.425)各组间MBP表达水平差异均无统计学意义(P0.05)。5.LPS+rh EPO组生后2周神经行为学评分(旷场试验、悬吊试验、斜坡试验、拒俘试验)与LPS+NS组、NS+rh EPO组及NS+NS组均无明显差别(F值分别为2.09、0.60、0.11、0.37)。结论1.宫内炎症致脑白质损伤新生大鼠内源性EPO m RNA及EPOR m RNA的表达明显增加。2.rh EPO可抑制脑白质损伤新生鼠的小胶质细胞和星形胶质细胞活化。3.rh EPO腹腔注射对脑白质损伤新生大鼠的远期神经行为改善作用尚不明确。
[Abstract]:Objective to establish a rat model of intracerebral leukopathy induced by intrauterine inflammation by intraperitoneal injection of lipopolysaccharide (LPS) in pregnant rats. Recombinant human erythropoietine rh EPO-rh EPOs were injected intraperitoneally to neonate rats with leukoencephalopathy. The neuroprotective effects of rh EPO on neonate white matter lesions were investigated by the detection of endogenous EPO-EPOR mRNA and CD68 / GFAPMA-MBP and the neurobehavioral score of rh EPO in the near and long term. To provide evidence for clinical application of rh EPO in the treatment of white matter lesions of preterm infants. Methods Thirty pregnant Wistar rats (gestational age 15 days) were randomly divided into experimental group and control group. The experimental group was injected intraperitoneally with LPS-300 渭 g/kg to make the animal model of white matter disease of premature infants, and the control group was treated with normal saline NS1 L / kg as control group. After birth, 30 neonatal rats were perfused with 4% paraformaldehyde and the left brain tissues were stained with HE to determine the white matter injury. The right brain tissue was used to detect the level of erythropoietin (EPO) and EPOR mRNA in corpus callosum by real-time quantitative RT-PCR. In the experimental group, 60 neonatal rats were randomly divided into LPS rh EPO group (n = 30) and LPS NS group (n = 30). In the control group, 60 neonatal rats were randomly divided into NS rh EPO group (n = 30) and NS group (n = 30). Ten newborn rats were randomly selected on the 3rd and 7th day after treatment. The brain tissues were stained with HE and CD68 GFAP and MBP were detected by immunofluorescence. At 2 weeks after birth, 10 rats were randomly selected for neurobehavioral score in each treatment group. Result 1. After intraperitoneal injection of LPS on the 15th day of gestation, the neonate rats were stained by HE with light staining of white matter around the ventricle, with sparse structure and reticular changes, while the white matter of control group was normal. 2. The expression of EPO mRNA and EPOR mRNA in corpus callosum of neonatal rats produced by intraperitoneal injection of LPS was significantly higher than that in NS group. On the 3rd day after birth, HE staining showed that the white matter around the ventricle of the neonatal rats in LPS rh EPO group was light stained, the structure was sparse, and the reticular changes were observed. The white matter structure of the newborn rats in the LPS NS group was sparse, which was more serious than that in the LPS rh EPO group. On the 7th day after birth, HE staining showed that the white matter structure around the ventricle was sparse in LPS rh EPO group, the white matter structure was sparse and reticular in LPS NS group, and the white matter structure was normal in NS rh EPO group and NS group 3 and 7 days after birth. The staining was clear. On the 3rd day after birth, the expression of CD68 in NS rh EPO group was significantly lower than that in LPS NS group. The expression of GFAP in NS group was significantly lower than that in LPS rh EPO group on 7 days after birth. The expression of GFAP in NS group was significantly lower than that in LPS group (F5. 121 P 0. 01). No statistical significance was found in P0.05. 5. The neurobehavioral score of 2 weeks after birth in LPS rh EPO group (open field test). There was no significant difference between the suspension test, slope test and captivity test) and the NS rh EPO group and NS group in LPS NS group. The F values of the suspension test, the slope test and the NS group were 2.09 卤0.60 and 0.110.37, respectively. Conclusion 1. Increased expression of endogenous EPO mRNA and EPOR mRNA in neonatal rats induced by intrauterine inflammation. 2. Rh EPO inhibited activation of microglia and astrocytes in neonatal rats with white matter injury The long-term neurobehavioral improvement in neonatal rats is unclear.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742

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