煤工尘肺对大鼠认知功能及海马区神经细胞凋亡的影响
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摘要
1.1目的
煤工尘肺(coal workers'pneumoconiosis,CWP)指煤矿工人长期吸入生产性粉尘所引起所引起肺部病变的总称。随着对煤工尘肺认识的逐渐深入,人们已发现其多种并发症可使病情恶化甚至死亡。煤工尘肺患者由于长期吸入生产性粉尘,使肺功能受损,造成慢性持续性缺氧。低氧血症会导致多器官的损害,尤其是需氧量较大的中枢神经系统。在缺氧与细胞凋亡关系研究中,发现缺氧对细胞凋亡具有诱导作用。神经细胞凋亡可引起脑功能障碍。现在人们普遍认为，线粒体是凋亡的执行者。同时线粒体在细胞凋亡调节中也十分重要,绝大多数蛋白分子通过线粒体起到调节凋亡的作用。由于煤工尘肺患者的慢性持续缺氧需较长时间才能导致认知障碍病等较严重的并发症,故在人体进行煤工尘肺并发认知障碍研究有一定的局限性,所以研制煤工尘肺动物模型对于阐明煤工尘肺致认知功能障碍及其机制非常重要。本实验旨在建立理想的煤工尘肺大鼠模型,研究煤工尘肺对大鼠海马神经元凋亡和认知功能的影响,为探讨煤工尘肺患者认知功能障碍的发生机制提供必要的实验依据。
1.2方法
1.2.1慢性持续性缺氧的煤工尘肺大鼠模型的制备
取健康3月龄雄性Wistar大鼠60只,所有大鼠先饲养一周,使其适应新环境。大鼠随机分为2组,即对照组和煤工尘肺(CWP)组,每组30只。用10%水合氯醛(300mg/kg)腹腔麻醉,然后行气管插管,将颗粒直径<5μm的煤粉尘配成50 g/L的生理盐水悬液,CWP组灌入煤尘生理盐水悬1 ml,对照组灌入1 ml生理盐水。所有大鼠正常饲料喂养,饮水与活动均不限。实验时间为16周。
1.2.2学习记忆能力测定
两组分别取十只于第4、8、16周通过水迷宫实验检测大鼠学习及记忆能力。①定位航行实验(place navigation):检验大鼠的学习记忆能力。②空间探索实验(spatial probe):检测大鼠的空间记忆能力。
1.2.3血气分析测定
于第4、8、16、周水迷宫实验后,腹腔麻醉,解剖开腹,用血气针取腹主动脉血0.5ml,作血氧分压的检测。
1.2.4形态学实验检测
实验结束时处死大鼠,取肺组织和脑组织做HE病理切片观察光镜下肺组织和脑组织结构的变化。取海马组织用TUNEL法检测神经细胞的凋亡情况。免疫组化法检测BCL-2/BAX蛋白表达变化情况。
1.3结果
1.3.1大鼠学习记忆测量结果
水迷宫实验结果显示,各组别学习记忆能力差别有统计学意义(p<0.05),CWP组4周、8周、16周差异有统计学意义(p<0.05)。正常组间比较不认为差异有统计学意义(p>0.05)。
1.3.2慢性持续性缺氧下的血气分析和肺组织病理切片结果
尘肺组大鼠动脉血氧分压与对照组相比明显降低,已经是慢性持续性低氧状态。染尘4周时可见炎细胞浸润增加,吞噬了煤尘的肺泡巨噬细胞增加及煤尘聚集形成的煤斑或结节增多,病灶肺气肿明显,染尘8、16周可见肺纤维化形成,符合煤工尘肺的病理特点。因此,从肺组织病理改变和慢性持续性缺氧状态来看此模型符合人类煤工尘肺的诊断标准。
1.3.3海马组织病理切片实验结果
海马组织病理形态学观察：光镜下,大鼠在染尘4周时,海马区变性神经元数量增多,胞体深染,排列散乱；在8周、16周时可见胶质细胞吞噬神经元,神经元坏死、减少,筛网状软化灶。采用TUNEL法检测海马区凋亡神经元。神经元胞核中有棕黄色颗粒的为阳性细胞。多组凋亡率指标之间的差别有统计学意义(p<0.05),正常组间比较差异不认为有统计学意义(p>0.05)。CWP组大鼠海马区凋亡神经元数目增多,明显高于对照组,两组比较有统计学差异(p<0.05)。
1.3.4大鼠海马区BCL-2/BAX免疫组化结果
光镜下见阳性细胞胞浆内出现棕黄色颗粒,各组均可见免疫组化染色的阳性表达细胞。但与对照组相比,CWP组BCL-2蛋白表达减少,BAX蛋白表达增加。经过析因分析可知两组蛋白表达差异有统计学意义,细胞凋亡率与BCL-2/BAX比值的相关性分析用spearman相关分析,二者呈负相关(p<0.01)。
1.4结论
(1)煤工尘肺大鼠学习记忆能力下降；
(2)慢性持续性缺氧可引起海马区凋亡调控蛋白表达发生变化,神经元凋亡率增加,可能为尘肺鼠认知障碍的发生机制之一。
Objective:Coal worker's pneumoconiosis (CWP) is the disease that is caused by the long-term inhalation of the production of dust pneumoconiosis of coal mine workers.
Because long-term inhalation of the production of dust pneumoconiosis, so that impaired lung function and causes a chronic continuous oxygen deficit.With the gradually deeper awareness about coal worker's pneumoconiosis，it has been found that in the progress of coal worker's pneumoconiosis process,it is prone to appear a variety of complications and secondary diseases resulting in the death. Chronic continuous oxygen deficit can cause multiple organ damage, especially the central nervous system. The relationship between hypoxia and apoptosis studies, found that the induction of hypoxia play the role of apoptosis. Neuronal apoptosis can cause brain dysfunction. It is now generally agreed that mitochondria is the executor of apoptosis. Mitochondria in apoptosis regulation is also important, the vast majority of protein molecules in regulating apoptosis through the mitochondria.Cognitive impairment caused by chronic continuous oxygen deficit of CWP happens after a long term, so it is difficult to conduct prospective study about the pathogenesis of coal worker's pneumoconiosis with cognitive impairment on human. Moreover, many intervening factors exist in clinical research, so experiments carried out on humans beings are limited. Therefore researches on CWP animals model would be useful to clarify the influence of CWP on cognitive and its payhogenesis. We look forward to establishing an suitable coal worker's pneumoconiosis model in rat, so that we can do some further researches on the influence of chronic continuous oxygen deficit on cognitive and the number of apoptosis neuron in hippocampus the possible pathogenesis. It will supply laboratory evidences for prevention and the clinical therapy coal worker's pneumoconiosis in patients with cognitive impairment.
Methods:1.Establishment of Chronic continuous oxygen deficit model of coal worker's pneumoconiosis in rat:60 healthy 3-month-old Wistar rats were chosen. All the rats were fed one week to adapt to the new environment before the experiement. rats were randomly divided into 2 groups, namely control group, coal worker's pneumoconiosis (CWP) group, every group 30.They were 10%chloral hydrate (300mg/kg) intraperitoneal anesthetized, then were tracheal intubated. CWP group rats were filled with lml coal dust suspension (the particle diameter <5μm of coal dust was made 50g/L of normal saline suspension) the control group rats were filled with 1 ml normal saline. All rats were fed with the normal feed, and their water and activities are not limited. Experimental time was16weeks.
2. The determination of the ability of lerning and memory (1) Morris water maze test learning scores (place navigation):evaluated the ability of lerning. (2) Morris water maze test memory scores(spatial probe)evaluated the ability of memory.
3. Blood gas analysis the two groups were respectively chosen 10 rats weeks intraperiton-eal anesthetized in4,8,16, were abdominal dissected, took 0.5ml of blood from the abdominal aorta with blood gas Lancets for the detection of blood oxygen partial pressure.
4.The determination of morphology:At the end of the experiment, rats were killed, took lung tissue and brain tissue to do HE staining to observe pathological changes of lung tissue and the structure of brain tissue. Observe the number of apoptosis neuron in hippocampus-(TUNEL) and the expression of BCL-2/BAX Protein of hippocampus.
Results
(1)The determination of the ability of lerning and memory①Morris water maze test learning scores:the escape latency in CWP rats was significantly longer than that in UC rats (P<0.05);②Morris water maze test memory scores. The number of times of crossing the platform in CWP group was significantly reduced than that in UC group (P<0.01). The percentage of time spent on crossing the target quadrant to the total swimming time in CWP group was also significantly decreased compared with that in UC group (P<0.01)
(2)The results of blood gas analysis and lung biopsyof chronic continuous oxygen defict model of CWP in rats:In the pre-experiment, the arterial partial pressure of oxygen in rats after experiment was 85.8-90.2mmHg, which comparing with the previous blood pressure was decreasing significantly, although did not meet the standard of hypoxemia,but was a state of chronic oxygen defict. After 4 weeks, inflammatory cells, alveolar macrophages with dust, coal-dust accumulation plaques or nodules, and emphysema increases.After 8and16 weeks, small areas of pulmonary fibrosis was formed which is consistent with the pathological characteristics of coal worker's pneumoconiosis. Therefore, from the pathological changes of the lung tissue and the state of chronic continuous oxygen deficit, this model was consistent with the diagnostic criteria of CWP in human.
(3)The results of the determination of morphology:from light microscope, in the control group and after two weeks, neurons and glial cells were normal. After 4 weeks, endothelial cell gap of small vessel wall increased and microglial cells aggregated.After 8,16 weeks, glial cells phagocytized neurons,neurons necrosis,reducedgrit-like mollification.Changes in the ultrastructure of hippocampus neurons and synapsis were obvious in CWP group as compared with the unhandled control group(2)the number of TUNEL positive nerve in hippocampus increased, CWP obviously higher than UC, the difference between the two groups is statistically significant(P<0.05)
(4)The expression of BCL-2/BAX of hippocampus:CWP was obviously decreased compared with that in UC group (p<0.05), spearman correlation analysis showed that bone cell apoptosis rate was negatively correlated withBCL-2/BAXratio (p<0.01).
Conclusion
(1) CWP can lead to cognitive impairment of the rats
(2) Chronic continuous oxygen deficit could lead the neuron poptosis in hippocampus maybe one of mechanism that chronic oxygen deficit hypoxia lead to cognitive impairment.
煤工尘肺(coal workers'pneumoconiosis,CWP)指煤矿工人长期吸入生产性粉尘所引起所引起肺部病变的总称。随着对煤工尘肺认识的逐渐深入,人们已发现其多种并发症可使病情恶化甚至死亡。煤工尘肺患者由于长期吸入生产性粉尘,使肺功能受损,造成慢性持续性缺氧。低氧血症会导致多器官的损害,尤其是需氧量较大的中枢神经系统。在缺氧与细胞凋亡关系研究中,发现缺氧对细胞凋亡具有诱导作用。神经细胞凋亡可引起脑功能障碍。现在人们普遍认为，线粒体是凋亡的执行者。同时线粒体在细胞凋亡调节中也十分重要,绝大多数蛋白分子通过线粒体起到调节凋亡的作用。由于煤工尘肺患者的慢性持续缺氧需较长时间才能导致认知障碍病等较严重的并发症,故在人体进行煤工尘肺并发认知障碍研究有一定的局限性,所以研制煤工尘肺动物模型对于阐明煤工尘肺致认知功能障碍及其机制非常重要。本实验旨在建立理想的煤工尘肺大鼠模型,研究煤工尘肺对大鼠海马神经元凋亡和认知功能的影响,为探讨煤工尘肺患者认知功能障碍的发生机制提供必要的实验依据。
1.2方法
1.2.1慢性持续性缺氧的煤工尘肺大鼠模型的制备
取健康3月龄雄性Wistar大鼠60只,所有大鼠先饲养一周,使其适应新环境。大鼠随机分为2组,即对照组和煤工尘肺(CWP)组,每组30只。用10%水合氯醛(300mg/kg)腹腔麻醉,然后行气管插管,将颗粒直径<5μm的煤粉尘配成50 g/L的生理盐水悬液,CWP组灌入煤尘生理盐水悬1 ml,对照组灌入1 ml生理盐水。所有大鼠正常饲料喂养,饮水与活动均不限。实验时间为16周。
1.2.2学习记忆能力测定
两组分别取十只于第4、8、16周通过水迷宫实验检测大鼠学习及记忆能力。①定位航行实验(place navigation):检验大鼠的学习记忆能力。②空间探索实验(spatial probe):检测大鼠的空间记忆能力。
1.2.3血气分析测定
于第4、8、16、周水迷宫实验后,腹腔麻醉,解剖开腹,用血气针取腹主动脉血0.5ml,作血氧分压的检测。
1.2.4形态学实验检测
实验结束时处死大鼠,取肺组织和脑组织做HE病理切片观察光镜下肺组织和脑组织结构的变化。取海马组织用TUNEL法检测神经细胞的凋亡情况。免疫组化法检测BCL-2/BAX蛋白表达变化情况。
1.3结果
1.3.1大鼠学习记忆测量结果
水迷宫实验结果显示,各组别学习记忆能力差别有统计学意义(p<0.05),CWP组4周、8周、16周差异有统计学意义(p<0.05)。正常组间比较不认为差异有统计学意义(p>0.05)。
1.3.2慢性持续性缺氧下的血气分析和肺组织病理切片结果
尘肺组大鼠动脉血氧分压与对照组相比明显降低,已经是慢性持续性低氧状态。染尘4周时可见炎细胞浸润增加,吞噬了煤尘的肺泡巨噬细胞增加及煤尘聚集形成的煤斑或结节增多,病灶肺气肿明显,染尘8、16周可见肺纤维化形成,符合煤工尘肺的病理特点。因此,从肺组织病理改变和慢性持续性缺氧状态来看此模型符合人类煤工尘肺的诊断标准。
1.3.3海马组织病理切片实验结果
海马组织病理形态学观察：光镜下,大鼠在染尘4周时,海马区变性神经元数量增多,胞体深染,排列散乱；在8周、16周时可见胶质细胞吞噬神经元,神经元坏死、减少,筛网状软化灶。采用TUNEL法检测海马区凋亡神经元。神经元胞核中有棕黄色颗粒的为阳性细胞。多组凋亡率指标之间的差别有统计学意义(p<0.05),正常组间比较差异不认为有统计学意义(p>0.05)。CWP组大鼠海马区凋亡神经元数目增多,明显高于对照组,两组比较有统计学差异(p<0.05)。
1.3.4大鼠海马区BCL-2/BAX免疫组化结果
光镜下见阳性细胞胞浆内出现棕黄色颗粒,各组均可见免疫组化染色的阳性表达细胞。但与对照组相比,CWP组BCL-2蛋白表达减少,BAX蛋白表达增加。经过析因分析可知两组蛋白表达差异有统计学意义,细胞凋亡率与BCL-2/BAX比值的相关性分析用spearman相关分析,二者呈负相关(p<0.01)。
1.4结论
(1)煤工尘肺大鼠学习记忆能力下降；
(2)慢性持续性缺氧可引起海马区凋亡调控蛋白表达发生变化,神经元凋亡率增加,可能为尘肺鼠认知障碍的发生机制之一。
Objective:Coal worker's pneumoconiosis (CWP) is the disease that is caused by the long-term inhalation of the production of dust pneumoconiosis of coal mine workers.
Because long-term inhalation of the production of dust pneumoconiosis, so that impaired lung function and causes a chronic continuous oxygen deficit.With the gradually deeper awareness about coal worker's pneumoconiosis，it has been found that in the progress of coal worker's pneumoconiosis process,it is prone to appear a variety of complications and secondary diseases resulting in the death. Chronic continuous oxygen deficit can cause multiple organ damage, especially the central nervous system. The relationship between hypoxia and apoptosis studies, found that the induction of hypoxia play the role of apoptosis. Neuronal apoptosis can cause brain dysfunction. It is now generally agreed that mitochondria is the executor of apoptosis. Mitochondria in apoptosis regulation is also important, the vast majority of protein molecules in regulating apoptosis through the mitochondria.Cognitive impairment caused by chronic continuous oxygen deficit of CWP happens after a long term, so it is difficult to conduct prospective study about the pathogenesis of coal worker's pneumoconiosis with cognitive impairment on human. Moreover, many intervening factors exist in clinical research, so experiments carried out on humans beings are limited. Therefore researches on CWP animals model would be useful to clarify the influence of CWP on cognitive and its payhogenesis. We look forward to establishing an suitable coal worker's pneumoconiosis model in rat, so that we can do some further researches on the influence of chronic continuous oxygen deficit on cognitive and the number of apoptosis neuron in hippocampus the possible pathogenesis. It will supply laboratory evidences for prevention and the clinical therapy coal worker's pneumoconiosis in patients with cognitive impairment.
Methods:1.Establishment of Chronic continuous oxygen deficit model of coal worker's pneumoconiosis in rat:60 healthy 3-month-old Wistar rats were chosen. All the rats were fed one week to adapt to the new environment before the experiement. rats were randomly divided into 2 groups, namely control group, coal worker's pneumoconiosis (CWP) group, every group 30.They were 10%chloral hydrate (300mg/kg) intraperitoneal anesthetized, then were tracheal intubated. CWP group rats were filled with lml coal dust suspension (the particle diameter <5μm of coal dust was made 50g/L of normal saline suspension) the control group rats were filled with 1 ml normal saline. All rats were fed with the normal feed, and their water and activities are not limited. Experimental time was16weeks.
2. The determination of the ability of lerning and memory (1) Morris water maze test learning scores (place navigation):evaluated the ability of lerning. (2) Morris water maze test memory scores(spatial probe)evaluated the ability of memory.
3. Blood gas analysis the two groups were respectively chosen 10 rats weeks intraperiton-eal anesthetized in4,8,16, were abdominal dissected, took 0.5ml of blood from the abdominal aorta with blood gas Lancets for the detection of blood oxygen partial pressure.
4.The determination of morphology:At the end of the experiment, rats were killed, took lung tissue and brain tissue to do HE staining to observe pathological changes of lung tissue and the structure of brain tissue. Observe the number of apoptosis neuron in hippocampus-(TUNEL) and the expression of BCL-2/BAX Protein of hippocampus.
Results
(1)The determination of the ability of lerning and memory①Morris water maze test learning scores:the escape latency in CWP rats was significantly longer than that in UC rats (P<0.05);②Morris water maze test memory scores. The number of times of crossing the platform in CWP group was significantly reduced than that in UC group (P<0.01). The percentage of time spent on crossing the target quadrant to the total swimming time in CWP group was also significantly decreased compared with that in UC group (P<0.01)
(2)The results of blood gas analysis and lung biopsyof chronic continuous oxygen defict model of CWP in rats:In the pre-experiment, the arterial partial pressure of oxygen in rats after experiment was 85.8-90.2mmHg, which comparing with the previous blood pressure was decreasing significantly, although did not meet the standard of hypoxemia,but was a state of chronic oxygen defict. After 4 weeks, inflammatory cells, alveolar macrophages with dust, coal-dust accumulation plaques or nodules, and emphysema increases.After 8and16 weeks, small areas of pulmonary fibrosis was formed which is consistent with the pathological characteristics of coal worker's pneumoconiosis. Therefore, from the pathological changes of the lung tissue and the state of chronic continuous oxygen deficit, this model was consistent with the diagnostic criteria of CWP in human.
(3)The results of the determination of morphology:from light microscope, in the control group and after two weeks, neurons and glial cells were normal. After 4 weeks, endothelial cell gap of small vessel wall increased and microglial cells aggregated.After 8,16 weeks, glial cells phagocytized neurons,neurons necrosis,reducedgrit-like mollification.Changes in the ultrastructure of hippocampus neurons and synapsis were obvious in CWP group as compared with the unhandled control group(2)the number of TUNEL positive nerve in hippocampus increased, CWP obviously higher than UC, the difference between the two groups is statistically significant(P<0.05)
(4)The expression of BCL-2/BAX of hippocampus:CWP was obviously decreased compared with that in UC group (p<0.05), spearman correlation analysis showed that bone cell apoptosis rate was negatively correlated withBCL-2/BAXratio (p<0.01).
Conclusion
(1) CWP can lead to cognitive impairment of the rats
(2) Chronic continuous oxygen deficit could lead the neuron poptosis in hippocampus maybe one of mechanism that chronic oxygen deficit hypoxia lead to cognitive impairment.
引文
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[11]SquirgiuD, Jeannette, GE.RonDK, etal.Quantification of swim patterns in the Morris Water maze. Behavior Researeh Methods,2000,32(2):134
[12]PengB, LiSW, KangH, Cognitive and emotional impairment in obstructive sleep apnea syndrome.ChinMedSeiJ.2004Dee:19(4):262-5.
[13]EI-Ad B, Lavie Effect of sleep apnea on cognition and mood. Int Rev Psyehiatry.2005 Aug; 17(4):277-82.
[14]AlemnaA, VehtaarHJ, denaaEH, Tetal.Insulin-likegrowthafetor-lnad cognitive ufnction inhealthy oldermne.JClinEndoerinolMetba,1999;84:471-475.
[15]林煜,陈俊抛,刘辉,等.学习记忆过程中海马SIOOβ和NOS表达的变化及其相关性.中国行为医学科学,2000,9：97-99
[16]De Jong D, Prins FA, Mason DY,et al.Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells [J]. Cancer Res,1994,54(1):256-260.
[17]Squire LR. Memory brain system and behavior.TINS,1988,11:17
[18]Somers V,Dyken M,Clary M,et al,Sympathetic neural mechanism in obstructive sleep aponea[J],J Clin Invest,1995,96:1897-1904
[19]Dimmeler S, Haendeler J, Nehls M, et al. Suppression of apoptosis by nitric oxide via inhibition of interleukin21 beta2converting enzyme (ICE)21ike and cysteine protease protein (CPP)23221ike proteases. J Exp Med,1997,185:6012607.
[20]Hla KM,Young TB, Bidwell T, et al,Sleep apnea and hypertension:A population-based study[J].Ann Intern Med,1994,120:382-388
[21]]Krajewski S,Mai JK, Krajiewski M,et al. Up2regulation of Bax protein levels in neurons following cerebral ischemia [J]. J Neurosci,1995,15(10):636426376.
[22]Isenmann S,Stoll G,Schroeter M,et al. Differential regulation of Bax,Bcl-2,and Bcl-Xproteins in focal cortical ischemia in the rat [J].BrainPathol,1998,8 (1):49262.
[23]段磊,王维平,张建生,等.人脑挫裂伤后神经细胞凋亡及凋亡相关基因Bcl-2/Bax的蛋白质表达.中国临床康复,2003,7(16)：2280～2281
[24]Peng B, Li SW, Kang H, et al. Cognitive and emotional impairment in obstructive sleep apnea syndrome. Chin Med Sci J,2004,19(4):262-265
[25]徐雁,李舜伟,黄席珍,等.阻塞性睡眠呼吸暂停综合征患者的认知障碍与胰岛素素样生长因子-1间的关系.中华医学杂志,2002,82(20)：1388-90
[26].徐斌,陈俊抛,林煜,等.去松果体对大鼠学习记忆及海马结构星形胶质细胞的影响.中国行为医学科学,2000,9：189-192
[27]lto I, Kawakami R, sakimura K, et al. Input-specific targeting Of NMDAreceptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses Neuropharma—Cology, 2000,39:943-951
[28]Sachdev PS, Brodaty H, Valenzuela M. The neuro-psychological profile of vascular cognitive impairment in stroke patient s [J]. Int Psychogeriat r,2003,15(Suppl 2):72273.
[29]贾建平.重视血管性认知障碍的早期诊断和干预[J].中华神经科杂志,2005,38(1)：426.
[30]Rockwood K. Vascular cognitive impairment and vas2cular dementia[J]. Neurol Sci,2002,23:2032204.
[1]Ishii-T, Moriyosh-K, Sugihara-H, et al. Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits.J-Biol-Chem.1993:268(4):2836-43
[2]Kumar A, Zou L, Yuan X, et al, N-methyl-D-aspartate receptors:tran-sient of NR1 /NR2A/NR2B subunits after traumatic brain injury in a rodent model. J Neurosci Res, 2002,67:781-786.
[3]Hrabetova S, Serrano P, Blace N, et al. Distinct NMDA receptor subpopulations contribute to long—term potentiation and long-term depression i-duction. Neurosci.2000.20: 81.
[4]Moriyoshi-K, Masu-M, Ishii-T, et al.Molecular cloning and Characterization of the rat NMDA receptor.Nature,1991:354(6348):31-7
[5]Nakanish-S,Molecular diversity of glutamate receptors and implications for brain function.Seienee,1992;258(5082):597-603
[6]Meguro-H, Mori-H, Arak-K, et al.Functional characterizatio of a heteromeric NMDA receptor channel expressed from cloned cDNAs.Nature.1992;357(6373):70-4
[7]Zukin-RS, Bennett-MVL. Alternatively spliced isoforms of the NMDAR1 receptor subunit.TINS.1995;18(7):306-313
[8]Buller-AL, Larson-HC, Sehneider-BE, et al.The molecular basis of NMDA receptor subtypes:native receptor diversity is predicted by subunit composition.J-Neurosei.1994;14(9):5471 84
[9]Sheng-M, Cummings-J, Roldan-LA, et al.Changing subunit composition of heteromeric NMDA receptors during development of rat cortex..Nature,1994, 10;368(6467):144-7
[10]Ishii-T, Moriyoshi-K, Sukihara-H, et al.Molecular characterization of thefamily of the N-methyl-D-aspartate receptor subunits.J-bio-ehem,1993,268(4):2836
[11]Chen-N, Luo-T, Raymond-LA.SubtyPe-dependence of NMDA receptor channel open probability.J-Neurosci.1999:19(16):6844-54
[12]Seherzer-CR, Landwehnneyer-GB, Kerne-JA, et al.Expression ofN-methyl-D-aspartate receptor subunit mRNAs in the human brain:Hippocampus and cortex.J-ComP-Neurol.1998:390(1):75-90
[13]Thompson-CL, Drewery-DL, Atkin-HD, et al.Immunohistochemical localization of N-methyl-D-aspartate receptor NR1, NR2A, NR2B andNR2C/D subunits in the adult mammalian cerebellum.Neurosci-Lett.2000;283(2):85-8
[14]林煜,陈俊抛,刘辉,等.学习记忆过程中海马SIOOβ和NOS表达的变化及其 相关性.中国行为医学科学,2000,9：97-99
[15]徐斌,陈俊抛,林煜,等.去松果体对大鼠学习记忆及海马结构星形胶质细胞的影响.中国行为医学科学,2000,9：189-192.
[16]Shmizu E, Tang YP, Rampon C, et al. NMDA Receptor-dependent synapticreinforce-ment as crucial process for memory consolidation. Science,2000,290: 1170-1174.
[17]lto I, Kawakami R, sakimura K, et al. Input-specific targeting Of NMD Areceptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses Neuropharma-cology,2000, 39:943-951
[18]Miyamoto Y. Yamada K, Noda Y, et al. Lower sensitivity to Stress andaltered monoaminergic neuronal fuInction in mice lacking the NMDA receptor epsihm 4 subunit. J Neurosci.2002.22:2335-2342.
[19]陈鹏慧,阮怀珍,吴席贵.缺氧及谷氨酸对大鼠下丘脑神经元NMDA通道的影响[J].第三军医大学学报,2001,23(4)：4292431.
[20]WilsonMA,TonegawaS.Synaptic plasticity,placecell and spatial memory:study with second generation knock outs[J].TINS,1997,20(3):102-106.
[21]刘燕强,顾景范.缺锌对大鼠脑组织游离氨基酸和突触膜N2甲基2D2天冬氨酸受体含量的影响[J].南开大学学报,2003,36(2)：21226.
[22]Horm EM,DillonGH,FanYP,etal.Developmentala spectsand mechanism so fratcaudal hypothal amic neuronal responses tohypoxia[J].JNeurophysiol,1999,81(4):194921959
[23]姚国恩,王景周,陈曼娥.血管性痴呆大鼠认知障碍的N2甲基2D2天冬氨酸受体机制[J].中国临床康复,2003,7(10)：149621498.
[24]Rampon-C, Tang-YP, Goodhouse-J, et al.Enrichment induces structuralChanges and recovery from nonspatial memory defieits in CA1 NMDAR1-knockout mice.Nat-Neurosci.2000;3(3):238-44
[25]Manabe-T, Aiba-A, Yamada-A, et al.Regulation of long-term potentiation by H-Ras through NMDA receptor phosphorylation.J-Neurosei.2000:20(7):2504-11
[26]Okabe-S, Collin-C, Auerbach-JM, et al.Hippocampal synaptic plasticity in mice over expressin ganemboniesu bunitof theNMD ArecePtor.J-Neurosci.1998:18(11):4177-88
[2]姜凤云,张建芳,赵玉洁等,291例煤工尘肺低氧血症的临床分析[J].中国工业医学杂志,1994,7(1)：24—25
[3]白莉,曹伟平,张映辉等.促凋亡蛋白Bid诱导肝细胞凋亡的机制.中国生物化学与分子生物学报,2004,20(5)：670-674.
[4]HaywardL, MantA, EylandA, etal Sleep disordered breathing and cognitive
function inaretire mentvill age PopulationAge11992:21:12121281
[5]Pardo OE, LesayA, ArcaroA, et al. Fibroblastfactor2-mediated translational control of IAPs blocks mitochondrial release of Smac/DIABLO and apoptosis in small cell lung cancer cells. MolCellBiol,2003,23 (21):7600-7610.
[6]Minn AJ,Velez P, Schendel SL,et al.Bcl-x(L) forms an ion channel in synthetic lipid membranes [J]. Nature,1997,3856 (6614):353-357.
[7]Ott M, Robertson JD, Gogvadze V,et al.Cytochrome c releasefrom mitochondria proceeds by a two-step process [J].Proc NatlAcad Sci USA,2002,99(3):1259-1263.
[8]彭黎明.六种细胞凋亡检测方法的比较.中华病理学杂志,1999,28：55257
[9]王新德.应重视成人痴呆的诊断和治疗.中华神经科杂志,1998,31(2)：133134
[10]周红,马忠森,王红丽等,TGFβ1和TGFβRI在煤工尘肺大鼠大鼠模型肺组织中的表达[J].吉林大学学报(医学版),2003,29(1)：152-154
[11]SquirgiuD, Jeannette, GE.RonDK, etal.Quantification of swim patterns in the Morris Water maze. Behavior Researeh Methods,2000,32(2):134
[12]PengB, LiSW, KangH, Cognitive and emotional impairment in obstructive sleep apnea syndrome.ChinMedSeiJ.2004Dee:19(4):262-5.
[13]EI-Ad B, Lavie Effect of sleep apnea on cognition and mood. Int Rev Psyehiatry.2005 Aug; 17(4):277-82.
[14]AlemnaA, VehtaarHJ, denaaEH, Tetal.Insulin-likegrowthafetor-lnad cognitive ufnction inhealthy oldermne.JClinEndoerinolMetba,1999;84:471-475.
[15]林煜,陈俊抛,刘辉,等.学习记忆过程中海马SIOOβ和NOS表达的变化及其相关性.中国行为医学科学,2000,9：97-99
[16]De Jong D, Prins FA, Mason DY,et al.Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells [J]. Cancer Res,1994,54(1):256-260.
[17]Squire LR. Memory brain system and behavior.TINS,1988,11:17
[18]Somers V,Dyken M,Clary M,et al,Sympathetic neural mechanism in obstructive sleep aponea[J],J Clin Invest,1995,96:1897-1904
[19]Dimmeler S, Haendeler J, Nehls M, et al. Suppression of apoptosis by nitric oxide via inhibition of interleukin21 beta2converting enzyme (ICE)21ike and cysteine protease protein (CPP)23221ike proteases. J Exp Med,1997,185:6012607.
[20]Hla KM,Young TB, Bidwell T, et al,Sleep apnea and hypertension:A population-based study[J].Ann Intern Med,1994,120:382-388
[21]]Krajewski S,Mai JK, Krajiewski M,et al. Up2regulation of Bax protein levels in neurons following cerebral ischemia [J]. J Neurosci,1995,15(10):636426376.
[22]Isenmann S,Stoll G,Schroeter M,et al. Differential regulation of Bax,Bcl-2,and Bcl-Xproteins in focal cortical ischemia in the rat [J].BrainPathol,1998,8 (1):49262.
[23]段磊,王维平,张建生,等.人脑挫裂伤后神经细胞凋亡及凋亡相关基因Bcl-2/Bax的蛋白质表达.中国临床康复,2003,7(16)：2280～2281
[24]Peng B, Li SW, Kang H, et al. Cognitive and emotional impairment in obstructive sleep apnea syndrome. Chin Med Sci J,2004,19(4):262-265
[25]徐雁,李舜伟,黄席珍,等.阻塞性睡眠呼吸暂停综合征患者的认知障碍与胰岛素素样生长因子-1间的关系.中华医学杂志,2002,82(20)：1388-90
[26].徐斌,陈俊抛,林煜,等.去松果体对大鼠学习记忆及海马结构星形胶质细胞的影响.中国行为医学科学,2000,9：189-192
[27]lto I, Kawakami R, sakimura K, et al. Input-specific targeting Of NMDAreceptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses Neuropharma—Cology, 2000,39:943-951
[28]Sachdev PS, Brodaty H, Valenzuela M. The neuro-psychological profile of vascular cognitive impairment in stroke patient s [J]. Int Psychogeriat r,2003,15(Suppl 2):72273.
[29]贾建平.重视血管性认知障碍的早期诊断和干预[J].中华神经科杂志,2005,38(1)：426.
[30]Rockwood K. Vascular cognitive impairment and vas2cular dementia[J]. Neurol Sci,2002,23:2032204.
[1]Ishii-T, Moriyosh-K, Sugihara-H, et al. Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits.J-Biol-Chem.1993:268(4):2836-43
[2]Kumar A, Zou L, Yuan X, et al, N-methyl-D-aspartate receptors:tran-sient of NR1 /NR2A/NR2B subunits after traumatic brain injury in a rodent model. J Neurosci Res, 2002,67:781-786.
[3]Hrabetova S, Serrano P, Blace N, et al. Distinct NMDA receptor subpopulations contribute to long—term potentiation and long-term depression i-duction. Neurosci.2000.20: 81.
[4]Moriyoshi-K, Masu-M, Ishii-T, et al.Molecular cloning and Characterization of the rat NMDA receptor.Nature,1991:354(6348):31-7
[5]Nakanish-S,Molecular diversity of glutamate receptors and implications for brain function.Seienee,1992;258(5082):597-603
[6]Meguro-H, Mori-H, Arak-K, et al.Functional characterizatio of a heteromeric NMDA receptor channel expressed from cloned cDNAs.Nature.1992;357(6373):70-4
[7]Zukin-RS, Bennett-MVL. Alternatively spliced isoforms of the NMDAR1 receptor subunit.TINS.1995;18(7):306-313
[8]Buller-AL, Larson-HC, Sehneider-BE, et al.The molecular basis of NMDA receptor subtypes:native receptor diversity is predicted by subunit composition.J-Neurosei.1994;14(9):5471 84
[9]Sheng-M, Cummings-J, Roldan-LA, et al.Changing subunit composition of heteromeric NMDA receptors during development of rat cortex..Nature,1994, 10;368(6467):144-7
[10]Ishii-T, Moriyoshi-K, Sukihara-H, et al.Molecular characterization of thefamily of the N-methyl-D-aspartate receptor subunits.J-bio-ehem,1993,268(4):2836
[11]Chen-N, Luo-T, Raymond-LA.SubtyPe-dependence of NMDA receptor channel open probability.J-Neurosci.1999:19(16):6844-54
[12]Seherzer-CR, Landwehnneyer-GB, Kerne-JA, et al.Expression ofN-methyl-D-aspartate receptor subunit mRNAs in the human brain:Hippocampus and cortex.J-ComP-Neurol.1998:390(1):75-90
[13]Thompson-CL, Drewery-DL, Atkin-HD, et al.Immunohistochemical localization of N-methyl-D-aspartate receptor NR1, NR2A, NR2B andNR2C/D subunits in the adult mammalian cerebellum.Neurosci-Lett.2000;283(2):85-8
[14]林煜,陈俊抛,刘辉,等.学习记忆过程中海马SIOOβ和NOS表达的变化及其 相关性.中国行为医学科学,2000,9：97-99
[15]徐斌,陈俊抛,林煜,等.去松果体对大鼠学习记忆及海马结构星形胶质细胞的影响.中国行为医学科学,2000,9：189-192.
[16]Shmizu E, Tang YP, Rampon C, et al. NMDA Receptor-dependent synapticreinforce-ment as crucial process for memory consolidation. Science,2000,290: 1170-1174.
[17]lto I, Kawakami R, sakimura K, et al. Input-specific targeting Of NMD Areceptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses Neuropharma-cology,2000, 39:943-951
[18]Miyamoto Y. Yamada K, Noda Y, et al. Lower sensitivity to Stress andaltered monoaminergic neuronal fuInction in mice lacking the NMDA receptor epsihm 4 subunit. J Neurosci.2002.22:2335-2342.
[19]陈鹏慧,阮怀珍,吴席贵.缺氧及谷氨酸对大鼠下丘脑神经元NMDA通道的影响[J].第三军医大学学报,2001,23(4)：4292431.
[20]WilsonMA,TonegawaS.Synaptic plasticity,placecell and spatial memory:study with second generation knock outs[J].TINS,1997,20(3):102-106.
[21]刘燕强,顾景范.缺锌对大鼠脑组织游离氨基酸和突触膜N2甲基2D2天冬氨酸受体含量的影响[J].南开大学学报,2003,36(2)：21226.
[22]Horm EM,DillonGH,FanYP,etal.Developmentala spectsand mechanism so fratcaudal hypothal amic neuronal responses tohypoxia[J].JNeurophysiol,1999,81(4):194921959
[23]姚国恩,王景周,陈曼娥.血管性痴呆大鼠认知障碍的N2甲基2D2天冬氨酸受体机制[J].中国临床康复,2003,7(10)：149621498.
[24]Rampon-C, Tang-YP, Goodhouse-J, et al.Enrichment induces structuralChanges and recovery from nonspatial memory defieits in CA1 NMDAR1-knockout mice.Nat-Neurosci.2000;3(3):238-44
[25]Manabe-T, Aiba-A, Yamada-A, et al.Regulation of long-term potentiation by H-Ras through NMDA receptor phosphorylation.J-Neurosei.2000:20(7):2504-11
[26]Okabe-S, Collin-C, Auerbach-JM, et al.Hippocampal synaptic plasticity in mice over expressin ganemboniesu bunitof theNMD ArecePtor.J-Neurosci.1998:18(11):4177-88