【作者】 宋岳涛；

【导师】 唐一鹏；

【作者基本信息】 北京中医药大学 ， 中西医结合基础—神经解剖学， 2004， 博士

【摘要】 研究脑缺血再灌注损伤的特点、规律和发生机制,对缺血性脑血管病的防治具有十分重要的意义。以往的研究大多数是单纯地从神经元或神经胶质细胞的角度来研究脑的缺血再灌注损伤,但神经元和胶质细胞是一个功能整体,应把二者有机地联系在一起进行研究。尤其是近年来缺血预适应已受到人们的关注,认为它是机体的一种内源性的保护机制。在这种脑缺血预适应中神经元发生缺血耐受现象,其中星形胶质细胞发挥了至关重要的作用。基于这样的认识,我们将从实验的角度来研究脑缺血再灌注损伤条件下星形胶质细胞与神经元的关系和中药的干预作用。由于神经元和星形胶质细胞的分离纯化培养技术为研究两者间的关系提供了强有力的手段,所以本实验研究了培养大鼠星形胶质细胞条件培养液对体外模拟脑缺血再灌注损伤神经元的作用和中药抗呆I号的影响,旨在探讨脑缺血再灌注损伤和脑缺血预适应的发生机理及其变化规律,并为中药防治缺血性脑血管病和新药研发提供更为科学和实用的实验依据。 本实验以离体培养的大鼠大脑皮层神经元和星形胶质细胞为研究对象,以体外模拟脑缺血再灌注损伤为实验条件,主要采用生化检测手段和免疫细胞化学技术,研究了体外模拟脑缺血再灌注损伤对培养神经元活性、星形胶质细胞分泌功能的影响以及星形胶质细胞条件培养液对受损神经元的作用和抗呆I号的影响,主要结果和结论如下:1.体外模拟脑缺血再灌注造成了培养神经元的损伤: 表现为:(1)受损神经元的活性降低,细胞存活率下降;(2)细胞培养液 LDH 的漏出率明显提高;(3)细胞死亡率显著上升;(4)NOS染色强阳性细胞数在缺血期和再灌注早期明显增多。 提示:体外模拟脑缺血再灌注造成了培养神经元的损伤,其损伤的原因可能是:(1)神经元线粒体受损,导致能量代谢障碍;(2)细胞膜结构的完整性遭到破坏,导致膜通透性改变引发破坏性的生化级联反应;(3)NO 及其衍生的毒性自由基产生增多,造成对神经元的毒性损伤。2.体外模拟脑缺血再灌注造成了培养星形胶质细胞的损伤: 表现为:(1)受损星形胶质细胞的活性降低,存活率下降;(2)受损星形胶质细胞分泌总体蛋白质的能力减弱。 提示:体外模拟脑缺血再灌注造成了培养星形胶质细胞的损伤。3.体外模拟脑缺血再灌注使受损神经元和星形胶质细胞的变化具有一定的规律性,可分:(1)细胞损伤期:从缺血开始至再灌注3h末;(2)功能代偿期:从再灌注3h末至再灌注18h末;(3)功能低下期:从再灌注18h末至再灌注36h末;(4)功能恢复期:从再灌注36h 末至再灌注72h 末。4.体外模拟脑缺血再灌注使星形胶质细胞分泌下列因子的能力增强,表达高峰分别为:(1)BDNF:再灌注36h末;(2)GDNF:再灌注3h末至再灌注18h末;(3)bFGF:再灌注3h末;(4)HSP70:再灌注48h末;(5)IL-6:从再灌注18h末至再灌注24h末。<WP=6>-2- 培养大鼠星形胶质细胞对拟脑缺血再灌注损伤神经元的作用和抗呆Ⅰ号的影响5.星形胶质细胞条件培养液(ACM)具有很强的营养活性: 表现为:(1)ACM 对受损神经元发挥其最大生物学效应的浓度为1:5;(2)再灌注18h后收集的ACM具有最高的营养活性;(3)ACM能使受损神经元的活性和存活率明显提高,使死亡率、细胞培养液LDH 的漏出率和NOS 强阳性细胞的表达量显著降低;(4)ACM 可明显增强受损神经元 NSE、bFGF 的受体(bFGF-R)和 bcl-2 的表达,降低受损神经元 bax和caspase-3的表达。 提示:(1)星形胶质细胞条件培养液对受损神经元具有很强的营养活性,从而推测星形胶质细胞在脑缺血预适应中发挥重要的作用;(2)bFGF-R的表达和bFGF的表达呈同步化,推测在脑缺血再灌注损伤中,神经营养因子和其受体的表达呈同步化,从而发挥其最大的生物学效应。6.抗呆Ⅰ号对受损的神经元和星形胶质细胞均具有保护作用: 主要表现为:(1)对受损神经元具有直接的保护作用,可提高受损神经元的活性和存活率,降低细胞培养液LDH的漏出率、细胞死亡率和NOS染色强阳性细胞的表达量;(2)对受损的星形胶质细胞也有直接的保护作用,可提高其活性、存活率以及培养液蛋白质的含量;(3)能增强受损星形胶质细胞分泌BDNF、GDNF、bFGF、HSP和IL-6的能力;(4)可明显增强受损神经元对NSE、bFGF的受体(bFGF-R)和bcl-2的表达,降低受损神经元对bax和caspase-3的表达;(5)抗呆I号可通过星形胶质细胞间接地保护和修复受损的神经元,因为在多数实验组中经抗呆I号作用的ACM(ACMK)的作用远大于ACM与抗呆I号联合应用(ACM+K)的作用,统计学上具有显著性差异。 提示:(1)中药抗呆Ⅰ号可能通过防止神经元的氧化磷酸化脱偶联而保护线粒体,防止脂质过氧化及通透性增加而保护细胞膜,抑制 NOS 活性的反应性增强而防止 NO 及其衍生的毒性自由基的损伤等途径而发挥对神经元的直接保护作用;(2)对受损的星形胶质细胞具有保护作用,且能增强其分泌神经营养因子、炎性细胞因子及应激反应蛋白的能力,从而间接地保护和修复受损的神经元;(3)对受损的神经元具有抗凋亡作用。 综上所述,体外模拟脑缺血再灌注使培养的神经元和星形胶质细胞造成损伤,在这种损伤条件下,机体会启动内源性的神经保护机制,使星形胶质细胞分泌具?更多还原

【Abstract】 It has very important meaning to the prevention and cure of cerebrovascular disease to studythe characteristic, disciplinarian and mechanism of the damage induced by cerebral ischemiareperfusion. The damage induced by cerebral ischemia reperfusion was investigated solely fromneuron or neuroglia in the past, however they are the functional whole and should be studiedtogether. In recent years particularly ischemia preconditioning(IPC) has beening concerned as ainherent sheltered mechanism by the people. During brain ischemia preconditioning(BIP) neuronsshow cerebral ischemia tolerance and astrocytes play a significant protective role. Based on thisviews ,we investigated the relation between astrocytes and neurons after ischemia andreperfusion ,and then studied the effect of Kangdai I through experiments.We researched the effectof cultured astrocyte conditioned medium on the damaged neurons induced by simulated cerebralischemia and reperfusion in Vitro and influence of Chinese herbal Kangdai I with the technique ofseparation and purification to cultured neurons and astrocytes. The aim is to probe into themechanism and regulation of cerebral ischemia and reperfusion injury and BIP ,so as to providemore scientific and reliable experimental evidences to the treatment of Chinese herbs forcerebrovascular diseases and the research & development of new medicine. In the experiment ,the neurons and astrocytes cultured cerebral cortex of rats wereresearched, based on simulated cerebral ischemia and reperfusion in vitro. We investigated theavtivity of cultured neurons and the secretory function of cultured astrocyte in the damage inducedby simulated cerebral ischemia and reperfusion in vitro, then studied the effect of astrocyteconditioned medium(ACM) on injured neurons and the influence of Kangdai I with biochemicalmethod and immunocytochemical technique .The main results and conclusions are as follows:1. Cultured neurons were damaged by simulated cerebral ischemia and reperfusion in vitro: Results: (1)The activity of the damaged neurons was decreased, the survival rate was descent. (2)The transudation rate of LDH in cultured medium increased obviously. (3)The mortality was increased significantly. (4) The strong positive cell count of NOS expression significantly was increased 4h afterischemia (Is-4h)and 3h after reperfusion (Rp-3h). Conclusion:The reason of neurons injury may be : (1) The injury of mitochondria results in disturbance of energetic metabolism. (2) The damage of cell membrane structure induces membrane infiltrative change to cause devastating continuous biochemical reaction.<WP=8>-4- 培养大鼠星形胶质细胞对拟脑缺血再灌注损伤神经元的作用和抗呆Ⅰ号的影响 (3) The increase of NO and its derived toxic free radicals cause the toxic harm on neurons.2. Cultured astrocytes were damaged by simulated cerebral ischemia and reperfusion in vitro: Results: (1)The activity of the damaged astrocyte was decreased, the survival rate was descent. (2)The ability of secreting total protein decreased. Conclusion: Cultured astrocytes was damaged in the model simulated cerebral ischemia andreperfusion in vitro.3. There were certain regularities in the change of damaged neurons and astrocytes induced bysimulated cerebral ischemia and reperfusion in vitro, it could be divided into: (1) Cell injury period: from the begin of ischemia to Rp-3h. (2) Functional compensation period: from the end of Rp-3h to Rp-18h. (3) Lower functional period: from the end of Rp-18h to Rp-36h. (4) Functional restoration period: from the end of Rp-36h to Rp-72h.4. The ability of astrocytes secretory was increased in the damage induced by simulated cerebralischemia and reperfusion in vitro, its high peak of expression was respectively as follows: (1) BDNF:The end of Rp-36h. (2) GDNF: from the end of Rp-3h to Rp-18. (3) bFGF:The end of Rp-3h. (4) HSP70:The end of Rp-48h. (5) IL-6: from the end of Rp-18h更多还原