【作者】 蔡建光；

【导师】 印大中；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 本论文是从整体动物水平和细胞分子水平研究脂质过氧化过程中的一个重要中间产物丙二醛（malondialdehyde,MDA）对SD大鼠学习记忆能力的影响及对培养的海马神经元胞内钙离子稳态的破坏作用。在整体动物水平上,用Morrise水迷宫试验研究了经侧脑室注射不同浓度MDA的SD大鼠在定位航行试验（place navigation test,PNT）和空间探索试验（spatial probe test,SPT）中的行为变化。与生理盐水处理组和/或空白对照组相比,发现大鼠在定位航行试验中寻找水下平台的逃避潜伏期（escape latent period,ELP）极显著地延长（P＜0.01）,在空间探索试验中120s时间内穿越水下平台次数显著减少（P＜0.05）,说明不同浓度的MDA导致了大鼠在空间学习和记忆能力的降低。运用透射电子显微镜（transmission electronicmicroscope,TEM）比较观察了生理盐水组/空白对照组和不同浓度MDA处理组大鼠海马CA1区神经元亚细胞结构和超微结构的变化,发现MDA处理后的大鼠CA1区海马神经元细胞内线粒体结构变形,线粒体内的折叠结构和附着在上面的嵴减少或者消失,突触连接结构如突触后致密物质（postsynaptic density material,PSD）的厚度减少,突触界面（curvature of synaptic interface,CSI）的曲度增加,突触后活性区（active zone,AZ）的长度缩短,但突触间裂隙的宽度（synapticcleft,SC）没有显著变化。说明MDA处理后的大鼠海马神经元结构受到了损伤,从而引起了其在学习能力和空间探索寻找水下平台的记忆能力的下降。在细胞分子水平上,MDA作用于原代培养的SD大鼠海马神经元,研究了其在光学显微镜下细胞形态学上的变化及对神经元细胞内游离Ca²⁺浓度变化的影响。结果表明:在光学显微镜下,可以清楚地显示随着体系中MDA浓度的升高,对培养的海马神经元的毒害作用增强以至于神经元表现出死亡和/或凋亡的形态特征。在MDA的浓度为1.0～1000μmol/L,随着浓度的升高和时间的延长,表现出对质膜Ca²⁺-ATPase(plasma membrane Ca²⁺-ATPase,PMCA)活性的抑制作用增强;在MDA浓度为1.0～1000μmol/L和30min的作用时间内,胞内游离Ca²⁺浓度([Ca²⁺]_i)也显著升高,并且出了一个早期的逐渐的升高（0～10min）和一个晚期的快速升高过程（15～30min）。用尼莫地平（nimodipine）阻断由L-型钙通道（L-VOCC）开放引起的外钙内流,可以抑制晚期胞内[Ca²⁺]_i升高,对早期胞内[Ca²⁺]_i升高没有影响;过量的Ca²⁺螯合剂EGTA,同样可以抑制晚期胞内[Ca²⁺]_i升高,而早期只在5min内有轻微地升高胞内[Ca²⁺]_i,推测晚期胞内[Ca²⁺]_i升高是由于质膜上L-VOCC开放,胞外Ca²⁺通过L-VOCC内流引起的;在体系中加入磷脂酶C（PL-C）活性抑制剂U73122,能抑制早期胞内[Ca²⁺]_i的升高,推测早期胞内[Ca²⁺]_i升高是由于MDA的作用激活了PL-C信号途径,使内质网（endoplasmic reticulum,ER）钙通道开放,ER钙库中的钙外流致胞内[Ca²⁺]_i升高;在体系中加入蛋白质激酶A（PKA）抑制剂H-89,该抑制剂对胞内[Ca²⁺]_i变化没有影响,推测cAMP/PICA信号途径可能没有参与MDA导致的胞内[Ca²⁺]_i变化。1995年,印大中教授和Brunk教授共同提出羰基毒化衰老学说。该学说认为,生物体代谢过程中产生的不饱和醛酮类物质（如MDA,4-HNE）是导致个体衰老和退行性疾病形成的核心物质和造成熵增性衰老改变的关键过程。本论文探讨了由于MDA的应激作用,在整体动物水平上导致SD大鼠学习能力和空间记忆能力的下降,在神经元的形态结构、亚细胞结构和超微结构上也观察到了由于MDA的作用导致的形态结构上破坏,并在分子水平上探讨了MDA作用下的海马神经元胞内钙稳态的破坏及可能的作用机制。更多还原

【Abstract】 The present study was performed in the whole animal level and cell molecular level to investigate the influences of carbonyl stress induced by malondialdehyde（MDA）,a typical intermediate of lipid peroxidation,on the learning and memory capability and intracellular Ca²⁺ hemostatsis in cultured hippocampal neurons of the SD rats.At the whole animal level,the place navigation test（PNT） and spatial probe test（SPT） were conducted using Morrise water maze test system after lateral cerebral ventricular injection of MDA into SD rats. Comparing to the physiological saline treated animals and/or blank control,it was found that the escape latent period（ELP） for searching the underwater platform was significant increased（P＜0.01） during the PNT and the incidence of crossing the underwater platform within 120s was decreased（P＜0.05） during the SPT.The experimental results demonstrated that the learning and memory capability were impaired underby different concentration of MDA.And also,the transmission electronic microscope（TEM） was applied for studying the hippocampal CA1 area neuronal ultra-structures of rats following treatment of different concentration of MDA.It was observed that the architectures of mitochondria in CA1 area neurons were deformed and their cristae were decreased or disappeared.The ultra-microstructures of synaptic junction including the thickness of postsynaptic density material（PSD）,the width of synaptic cleft（WSC）,the curvature of synaptic interface（CSI） and the length of active zone（LAZ） were also altered in different severity.These results suggested that remarkable decrease of learning and memory capability were resulted from the alteration of hippocampal neuronal architectures and their synptic junction after MDA stress.At the cellular and molecular levels,the morphological alterations were conducted using the light microscope and the Ca²⁺ hemostasis was performed in cultured hippocampal neurons.The microphotographic study clearly demonstrated that the hippocampal neurons became gradually damaged following exposure to different concentrations of MDA.Further study indicated that the plasma membrane Ca²⁺-ATPase （PMCA） activity was inhibited by MDA in a concentration（1.0～1000μMol/L） and time（30 min） dependent manner.The supplementation of 100μMol/L MDA was found to cause a notable early phase increase of [Ca²⁺]_i in hippocampal neuron cultures followed by a more pronounced late phase elevation of[Ca²⁺]_i.Such effect of MDA was prevented by the addition of nimodipine,an inhibitor of L-type calcium channel or by an extracellular Ca²⁺ chelator EGTA.The identification of the calcium signalling pathways were studied by applying U73122,an inhibitor of PL-C,and H-89,an inhibitor of protein kinase A（PKA）,showing the involvement of PL-C/IP3 pathway but not the PKA/cAMP pathway. These results suggested that MDA-related carbonyl stress caused damages of rat hippocampal neurons by triggering Ca²⁺ influx and influencing Ca²⁺ homeostasis in cultured neurons,and also MDA may act as a signalling molecule regulating Ca²⁺ release from intracellular stores.In 1995,the aging theory of "carbonyl stress"was brought out by Professor Yin Dazhong and Brunk.This theory suggests that unstatured aldehydes and ketons（such as MDA and 4-HNE）,which were originated from metabolic processes of orgnisms,were the critical processes and crucial materials of biological aging and different types of degenerative diseases.The present study probed the possible"carbonyl stress" mechnisms resulting in impairment of learning and memory capability of SD rats under carbonyl stress at whole animal level.A significant damage was displayed of the structure of mitochondira and synaptic junction at cellular and sub-cellular levels.And also,the possible mechanisms of intercellular Ca²⁺ homeostasis disrupted following MDA stress were investigated at molecular level.更多还原