【作者】 马静；

【导师】 朱薇薇；

【作者基本信息】 山东大学 ， 儿科学， 2008， 硕士

【摘要】 目的围产期窒息所致的新生儿缺氧缺血性脑病（hypoxic-ischemicencephalopathy,HIE）是严重威胁新生儿生命和健康的常见病之一,死亡率和致残疾率高,关键原因在于中枢神经损伤后缺乏再生的能力,一般对症治疗不能有效改善其症状及预后。传统的观点认为中枢神经损伤后不能再生,而近年的研究发现阻止中枢神经损伤后再生的重要原因是中枢神经的内环境中存在抑制神经再生的因子,目前已确认的中枢神经髓鞘来源的抑制因子至少有以下三种:Nogo-A、髓磷脂相关糖蛋白（myelin-associated glycoprotein,MAG）和少突胶质细胞-髓磷脂糖蛋白（oligodendrocyte-myelin glycoprotein,OMgp）,其中Nogo-A是其中抑制作用最强的一种,其羧基端两个跨膜区之间存在一个66个氨基酸的结构域（Nogo-66）,这三种抑制因子通过相同的受体（Nogo receptor,NgR）发挥抑制神经再生的作用。NEP1-40是针对Nogo-66氨基序列设计的NgR竞争性拮抗剂,能阻断中枢神经抑制因子与NgR的结合。神经节苷脂为近年常用于治疗HIE的药物,有维持细胞内外离子平衡、防止细胞内Ca²⁺积聚及对抗兴奋性氨基酸的神经毒性等作用。本研究通过制备新生大鼠HIBD标准模型,对HIBD后大鼠分别腹腔注射NEP1-40及单唾液酸四己糖神经节苷脂钠（GM-1）,观察各组大鼠脑重的增长、左右脑重差值、脑组织病理变化及Nogo-A mRNA的表达,探讨Nogo-A在新生大鼠HIBD中的作用,并观察NEP1-40与GM-1对大鼠Nogo-A表达的影响及对神经损伤后的保护作用,为进一步研究新生儿缺氧缺血性脑病有效治疗方式提供动物实验依据。方法将100只7日龄新生大鼠随机分为空白对照组、假手术组、缺氧缺血性脑损伤（HIBD）模型组、NEP1-40治疗组及GM-1治疗组,以上各组均在6h、24h两时间点采集标本,每组10只。HIBD组及治疗组参照Rice法制备标准HIBD模型,治疗组于造模后即刻腹腔注射NEP1-4010mg/kg、GM-120mg/kg,空白对照组、假手术组与模型组腹腔注射生理盐水0.25ml/kg。各组动物分别处理后于6h、24h两时间点断颈处死。将脑组织标本固定于30%蔗糖、4%多聚甲醛溶液中静置72h,待下沉后进行冰冻切片,HE染色观察各组大鼠脑组织病理变化,用原位杂交的方法观察Nogo-A mRNA的表达,40倍镜下计数大鼠脑组织Nogo-A mRNA阳性细胞数目。结果①HIBD组两时段,Nogo-mRNA表达均高于同时段空白组及假手术组:HE染色示脑组织神经元水肿、尼氏小体溶解坏死,脑实质内有散在的凝固性坏死,脉络丛上皮局部缺失。②NEP1-40治疗组能抑制HIBD后mRNA的表达,与同时段模型组对比有显著统计学意义;神经元轻度水肿,未见溶解坏死的尼氏小体,脉络丛上皮部分缺失,毛细血管基本正常。③神经节苷脂治疗组mRNA的表达在6h时与HIBD组无明显差异,在24h时较HIBD组低但高于空白组。24h神经元细胞水肿较模型组减轻,可见少量坏死尼氏小体,脉络丛上皮连接基本正常。结论Nogo-A mRNA在缺氧缺血性脑损伤后表达显著增高,其编码的Nogo-A可抑制中枢神经损伤后的再生,NEP1-40能拮抗这一作用,能减轻细胞水肿、改善局部血流供应,从而可能在促进缺氧缺血性脑损伤后神经再生中起到重要作用。神经节苷脂GM-1在缺氧缺血性脑损伤后也可部分抑制Nogo-A mRNA的表达,有稳定细胞膜、减轻细胞水肿的作用。更多还原

【Abstract】 Objective The hypoxic-ischemic encephalopathy（HIE）caused by asphyxia in peripartum is a serious disease in newborn infants,with a high disability rate and mortality rate.The most important question is that the center nerve system was not able to regenerate after injury.The traditional symptomatic treatment can not improve the prognosis effectively.But lots of studies in recent years discovered that there were factors caused the failure of regeneration.At present,there were at least three factors were found in center neural myelin sheath to produce marked effects:Nogo-A,myelin-associated glycoprotein（MAG）, oligodendrocyte-myelin glycoprotein（OMgp）.Nogo-A was able to produce a most important effect.There was a structural domain composed by 66 amino acids in carboxyl terminus.All the three inhibiting factors effected by combined with nogo receptor.NEP1-40 was designed to aim directly at the structural domain,and it can antagonize the combination competitively. Ganglioside was commonly used to treat HIBD,it can stabilize the cellular membrane,ease cellular edema,prevent the accumulation of Ca²⁺, antagonize the neurotoxicity of excitatory amino acids.In this study, HIBD model of newborn rats were prepared,then NEP1-40 and GM-1 were injected in their belly cavity respectively.After got the brain tissue, the weights of brains,the pathological change,the contents of Nogo-A mRNA were observed to investigate the function of Nogo-A,the effects of NEP1-40 and GM-1 in the newborn rats with hypoxic ischemic brain damage （HIBD）. Methods 100 rats were randomly divided into 10 groups:normal control group,sham operated group,HIBD model group,NEP1-40 treatment group and GM-1 treatment group after HIBD at 6h and 24h.HIBD model groups were prepared by Rice method,NEP1-40（10mg/kg）and GM-1（20mg/kg）were injected in the belly cavity of two treatment groups respectively,while,normal sodium（0.25ml/kg）were injected in the belly cavity of the normal control groups,sham operated groups,and HIBD model groups.The tissues were fixed in 30%sugar solution with 4%polyoxymethylene.Then the tissues were prepared by the frozen microtome section.The pathological changes were observed by HE drum dyeing,and the contents of Nogo-A mRNA were detected by the method of in situ hybridization.Results①The expression of Nogo-A mRNA in HIBD groups was higher than that in control groups at both time points.In the HE section,The nerve cells were edematous,Nissl’s bodies were necrotic,and the endothelial cells of the choroid plexus were not complete.②In NEP1-40 groups,the expression of Nogo-A mRNA were lower than that in HIBD groups at both time points.The nerve cells were edematus lightly,and the endothelial cells and capillaries of the choroid plexus were mostly normal.③There was no great difference between the GM-1 group and HIBD group when they were treated at the 6th hour.And for the 24th-hour-group,the expression in GM-1 group was lower than that of HIBD group but higher than that of normal control group.The cellular edema was eased than in HIBD model groups, and the choroid plexus were incomplete partly.Conclusion Nogo-A mRNA increased obviously in the newborn rats with HIBD. Nogo-A encoded by the mRNA can inhibit the regeneration of central nerve after being injured,and NEP1-40 can antagonize the function,and be able to ease cellular edema,to improve the regional flow supply of the injured nerve,thereby it can promote the regeneration.GM-1 was also able to antagonize the expression of mRNA,to stabilize the cellular membrane, to ease cellular edema of the injured nerve and to accelerate the regeneration of new nerve.更多还原