【作者】 车菊华；

【导师】 王苏平；

【作者基本信息】 大连医科大学 ， 神经病学， 2008， 硕士

【摘要】 研究背景和目的:急性一氧化碳中毒(acute carbon monoxide poisoning,ACMP)是较常见的职业性及生活性中毒,可造成中枢神经系统(centeral neverous system,CNS)损伤。部分患者急性中毒症状消失以后,经过数天或数周表现正常或接近正常的假愈期,会出现以痴呆为主的精神神经症状称为急性一氧化碳中毒迟发性脑病( delayed encephalopathy after acute carbon monoxide poisoning,DEACMP),DEACMP患者颅脑影像学上白质脱髓鞘显著[1-4]。假愈期的存在给DEACMP的早期诊断带来困难,给有效防治带来困难,给社会家庭造成很大负担。Nogo-A蛋白是神经元轴突生长抑制因子之一,主要表达于CNS的少突胶质细胞[5],已证实Nogo-A可触发神经元轴突抑制、生长锥崩溃、阻止神经纤维散布,其受体(Nogo- receptor ,NgR)广泛分布于多种神经元,还存在于胶质细胞间隙缝连接处,提示NgR可通过隙缝连接来调节胶质细胞间的联系,但少突胶质细胞本身无NgR分布[6]。这些使得Nogo-A及NgR成为限制神经再生和轴突损伤修复的关键因素[7]。Nogo-A是否参与了ACMP脑损害、DEACMP的发病机制尚未见报道。鉴于Nogo-A蛋白及NgR的分布特点,及DEACMP患者颅脑影像学上白质脱髓鞘突出,我们推测Nogo-A蛋白及NgR与ACMP脑损害,尤其与DEACMP关系密切。内源性CO是重要的气信使分子之一,具有传递细胞间信息、调节细胞功能的作用[8],CO由血红素氧合酶(hemeoxygenease,HO)代谢产生的,HO抑制剂锌原卟啉-9(Zinc Porphyrin IX,ZnPPIX)可抑制CO生成,已用于研究内源性CO的神经内分泌调节作用[8]。作为体内合成内源性CO的唯一酶系统,HO-CO系统日益受到关注,其对Nogo-A及NgR的影响目前亦未见报道。本实验中,排除缺氧影响,我们用外源性CO直接处理体外培养少突胶质细胞,并用ZnPPIX抑制了HO活性,观察少突胶质细胞Nogo-A在mRNA及蛋白质水平的表达情况,以期探讨Nogo-A在ACMP脑损害和DEACMP中可能存在的病理生理机制,为研究ACMP脑损害和DEACMP的发病机制和治疗方面提供一个新的实验依据。方法:(1)取新生2天SD大鼠的视神经,采用组织块培养法,用DMEM/F12化学限定培养液培养、纯化少突胶质细胞。细胞免疫化学检测少突胶质细胞髓鞘碱性蛋白(Myelin basic protein,MBP)表达,鉴定少突胶质细胞,并计算阳性率。(2)在排除缺氧的影响下,1%CO处理少突胶质细胞,采用RT-PCR及细胞免疫组化观察6h、24h、48h少突胶质细胞Nogo-AmRNA及其蛋白表达。(3)取Nogo-AmRNA表达最高的时间点,预先加入ZNPP-IX抑制HO活性,1%CO处理少突胶质细胞,检测Nogo-AmRNA及其蛋白。结果:1、组织块24h开始贴壁,48h-72h可见神经组织边缘游出少量细胞,主要为圆形。9-10d左右细胞基本从组织块中游离出来,平铺于皿底。11d左右获得的细胞胞体基本为呈圆形或多角形,直径约6-10μm,细胞核较大,胞质少,突起丰富,交织成网。细胞细胞免疫化学染色MBP蛋白阳性,95%以上为阳性细胞。2、RT-PCR检测Nogo-AmRNA表达;(1)对照组:6h、24h、48h均有Nogo-A mRNA表达分别为:0.733±0.034、0.705±0.027、0.717±0.04,且未见其表达随时间发生明显变化;CO组:6h、24h、48h Nogo-AmRNA表达分别为: 1.042±0.015、1.304±0.008、0.937±0.005,与对照组相比有统计学差异(P<0.05)。(2)ZNPP-IX组与单纯CO处理组( COZN)相比Nogo-AmRNA增加,值为: 1.454±0.041、1.278±0.032,(P<0.01),有统计学差异。3、细胞免疫化学检测Nogo-A蛋白表达:Nogo-A表达于少突胶质细胞的胞膜和胞浆中,为棕褐色。免疫细胞化学染色图像分析结果显示:(1)Control组6h、24h、48h均有Nogo-A蛋白表达,未见其表达随时间发生明显变化,累计光密度值为:2836.74±94.30;2761.16±75.80 ; 2780.16±95.46 ; CO组6h、24h、48h累计光密度值为:4087.20±79.28;7240.40±63.25;3449.90±93.46;CO组各时间点累计光密度值与Control组相比有统计学差异(P<0.05)(3)ZNPP-IX组:ZNPP-IX干预后与单纯CO处理组(COZN)相比Nogo-A蛋白表达增加,有统计学差异(P<0.05),累计光密度值分别为:7159.41±97.32;9880.76±105.81。结论:(1)在排除缺氧的影响下,外源性CO诱导体外培养少突胶质细胞Nogo-A mRNA及蛋白表达增加。(2)在排除缺氧的影响下,外源性CO诱导体外培养少突胶质细胞Nogo-A mRNA表达增加时,HO-CO系统对Nogo-A mRNA及蛋白表达有抑制作用。更多还原

【Abstract】 Background and purpose: Acute carbon monoxide poisoning (ACMP) is the common occupational poison in our life.It can cause the injury of central nervous system (CNS).After acute poisoning symptoms disappeared , some patients will present normal or near normal for a few days or weeks ,It called the false period.Then They present mainly the dementia symptoms ,which known as the delayed encephalopathy after acute carbon monoxide poisoning, DEACMP, Brain MRI of patients with DEACMP ,the white matter is demyelinating significantly[1-4]. Because of the false period, it is difficult to diagnose DEACMP in forepart accurately. It is difficult to effectively prevent and control, and bring a great burden on the family which has the patient. Nogo-A is one of the nervous growth inhibitors in CNS.It mainly expressed by oligodendrocytes which are in the CNS[5].It has been confirmed that Nogo-A can trigger the growth cone collapse , inhibite the regrowth of nerve axons,and block prevent the Spread of the nerve fibers , and its receptor (NgR) widely distributes in lots of neurons,which immune electron microscopy studies show that NgR has immune response to glial cells and also exist in the seam connection in glial cells, NgR which is in the seam connection between the glial cells may regulate their signal transmit,but there is no NgR in oligodendrocytses[6].All those make Nogo-A and it’s receptors become the key factor to restricte the regrowth of nerve axons and prevent the regeneration of CNS from it’s injury [7] .whether Nogo-A was involved in the ACMP’s brain damage and DEACMP the mechanism or not ,which has not been reported. In view of the distribution of Nogo-A and its receptors, also the white matter demyelinated prominently on DEACMP patients’s brain MRI, we speculate that Nogo-A and it’s receptor system have some contributions to the ACMP, especially to the DEACMP. Endogenous CO is one of the important elements of the air courier. It can transfer the information between cells and regulating the function of cells . CO mainly produce by the metabolism of the Heme oxygenase ( HO).Zinc Porphyrin IX (ZnPPIX) is the inhibitor of HO, it can inhibit the produce of endogenous CO, and it has been used to study the neuroendocrine regulator function of the endogenous CO[8]. As the only one system of synthesizing endogenous CO,the HO-CO system has been pay more attention.In addition, CO as a gas messenger molecules in brain, its relationship whit Nogo-A and NgR also has not been reported in the present. In our study, after excluding the effect of hypoxia,we treat oligodendrocytes with 1%CO directly, at the same time we use ZnPPIX inhibit the produce of the endogenous CO ,and then observed the expression of Nogo-AmRNA and its protein. In order to discusss the potential pathophysiological function of Nogo-A and the HO-CO system in ACMP and DEACMP, and provid a new experimental clue for studying the pathogenesis and treatment of ACMP and DEACMP.Methods: (1) The optic nerves of 2 days’newborn SD rats.Optic nerve tissues were cultured directly and DMEM/F12 chemically defined culture medium was used to culture and purify oligodendrocytes. We detect the expression of the myelin basic protein (MBP) in oligodendrocytses by immunocytochemical examination.(2) Excluding the effect of hypoxia, we treat oligodendrocytes with 1% CO directly, and detect the expression of Nogo-A mRNA and Nogo-A protein of 6h, 24h, 48h by RT-PCR and immunohistochemical. (3) Select the time when the Nogo-A mRNA express into the peak value, and pre-add ZNPP-IX in medium in order to inhibit the produce of endogenous CO, detect the expression of Nogo-A mRNA and protein by RT-PCR and immunohistochemical.Results: 1、After 24 hours,tissue is adherenting to Petri dish,After 48-72hours ,the edge of the nerve tissue has a small number of rotundate and cells travelling out. About 9-10 days later all the cells of optic nerves can almostly traveled out and overspread the dish. In the 11days, the cells growth into a round and polygonal shape, the diameter is about 6-10um. The cells have larger nuclei and less cytoplasm. We bserved more than 95 percent cells are positive by immunocytochemical examination.2、Detect the expression of Nogo-AmRNA by RT-PCR:(1) The expression of Nogo-A mRNA of the control group (control): 6h, 24h, 48h are:0.733±0.034, 0.705±0.027,0.717±0.04, and there is no significantly change; the expression of Nogo-AmRNA of the CO treated group compared with the control group has increased at each time point: 6 h (1.042±0.015 vs 0.733±0.034, P <0.05); 24h (1.304±0.008 vs 0.705±0.027,P <0.05);48h (0.937±0.005 vs 0.717±0.04, P <0.05)。(2) The ZNPP-IX group compared with COZN,the expression of Nogo-AmRNA has significantly increased(1.454±0.041 vs 1.278±0.032, P <0.01). 3、The expression of Nogo-A protein of each group has the same changes with the expression of Nogo-AmRNA. The Cumulative optical density of each group the control group 6h, 24h, 48h are 2836.74±94.30,2761.16±75.80,2780.16±95.46;the CO group 6h,24h,48h are4087.20±79.28,7240.40±63.25,3449.90±93.4,compared with the control group has significantly change(P<0.05);the ZNPP-IX group is 9880.76±105.81, compared with the COZN group has significantly change(P<0.05)。Conclusion: (1) Excluding the influence of hypoxia,exogenous CO can increase the expression of Nogo-A mRNA and its protein of oligodendrocytes which is cultured in vitro.(2)Excluding the influence of hypoxiawhen exogenous CO increases the expression of Nogo-A mRNA and its protein of oligodendrocytes,the HO-CO system can inhibit the expression of Nogo-A mRNA and its protein.更多还原