银杏叶提取物及磷酸肌酸钠对A β25-35致PC12细胞凋亡保护作用机制研究
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摘要
目的:本文通过淀粉样蛋白β(Aβ)25-35诱导PC12细胞凋亡,建立阿尔茨海默病的细胞膜型,并给予银杏叶提取物(EGB)及磷酸肌酸钠(PCr)进行干预,了解二者是否对Aβ25-35所致的神经元凋亡有保护作用。
方法:取对数生长期PC12细胞,细胞计数后,以104/mL的密度接种于96孔板中,培养24小时后,半量换液,并加入不同浓度的Aβ25-35(A:0μmol/L (对照组)、B:10μmol/L、C:20μmol/L、D:30μmol/L、E:40μmol/L)。培养24小时后测得细胞存活率,应用SPSS软件选择方差分析法进行统计分析,发现在Aβ25-35浓度为30μmol/L时对PC12细胞的损伤效果明显。按相同条件进行细胞培养24小时后加入Aβ25-35,浓度为30μmol/L,12小时后,按下面分组方法分别加入银杏叶提取物及磷酸肌酸钠:银杏叶提取物A:Aβ0umol (对照组)、B:Aβ30umol/L、C:Aβ30umol/L +EGB100mg/L、D:Aβ30umol/L+EGB200mg/L、E:Aβ30umol /L +EGB400mg/L,磷酸肌酸钠组A:Aβ0umol/L (对照组)、B:Aβ30umol /L、C:Aβ30umol/L+PCr10mmol/L、D:Aβ30umol/L + Cr20mmol/L、E:Aβ30umol/L+PCr 30 mmol /L。培养12小时后,CCK-8法测细胞存活率,统计分析后得出,银杏叶提取物组200mg/L,磷酸肌酸钠组20mmol/L对细胞保护作用明显。按上述方法用Aβ25-35浓度为30μmol/L损伤PC12细胞,12小时后予银杏叶提取物组(200mg/L), PCr组(20mmol/L)保护细胞,培养12小时后,用线粒体膜电位特异荧光探针JC-1标记PC12细胞,启动共聚焦显微镜,选择488nm氩离子激光和543nm氦氖激光作为激发光,选择530±15 nm和615±30 nm通道作为接收通道,启动计算机运行Lasersharp2000软件,扫描图象,最后将图象在Laserpix软件中进行处理。同时将浓度为104/mL的细胞,以600转/分的速度进行细胞甩片后,按TUNEL凋亡试剂盒的操作步骤检测细胞凋亡,计算细胞凋亡率,采集图像。采用SPSS统计软件进行统计学分析。
结果:在Aβ25-35浓度为30μmol/L时,对PC12细胞损伤效果明显,与0μmol/L,10μmol/L ,20μmol/L组有显著差异( P < 0.01 ),而和40μmol/L组,差异不明显(P> 0.01)。以不同浓度的银杏叶提取物及磷酸肌酸钠进行干预后,测量OD值,采用方差分析法得出EGB200mg/L组与Aβ30umol /L,EGB 100 mg/L组差异明显(P<0.01),而和400 mg/L组及Aβ0 umol /L组无明显差异(P>0.01),PCr 20 mmol/L与Aβ30umol /L,PCr 10 mmol/L组差异明显(P< 0.01 ),和PCr 30mmol/L组无明显差异。线粒体膜电位示30umol/L的Aβ处理PC12细胞(简称为Aβ组,下同)24小时后,红色荧光强度明显降低,绿色荧光强度明显增强,即线粒体膜电位(MMP)降低。而以Aβ30umol/L + EGB200mg/L组处理PC12细胞(简称为银杏组,下同)及Aβ30umol/L+PCr 20mmol/L组同时处理PC12细胞(简称为磷酸肌酸钠组,下同)后,红色荧光强度明显增强,绿色荧光强度明显降低,即线粒体膜电位(MMP)升高。Aβ组(30umol/L Aβ)、EGB组(Aβ30 umol/L + EGB 200mg/L)及PCr组(Aβ30umol/L + PCr 20mmol/L)均可见TUNEL阳性细胞,即凋亡细胞,PO-D标记的凋亡细胞显绿色荧光;而以DAB显色,凋亡细胞核被染成棕色。Aβ组细胞凋亡率(63%)高于银杏组(21%)及磷酸肌酸组(22%)(p<0.01)。
结论:1.浓度为30umol/L的Aβ25-35可诱导PC12细胞凋亡,建立AD的细胞膜型。2.浓度为200mg/L的银杏叶提取物及20mmol/L的磷酸肌酸钠,通过提高PC12细胞的线粒体膜电位水平,抑制细胞凋亡,提高细胞存活率,从而对Aβ25-35损伤的PC12细胞起到一定的保护作用。
Objective: This experiment uses amyloidβ(Aβ) 25-35 to induce apoptosis in PC12 cells, establishing the cell type of Alzheimer’s disease, and then provides Ginkgo biloba extract and creatine phosphate sodium to intervene. To explore whether the two play protective roles in neuronal apoptosis induced by Aβ25-35.
Materials and methods: Choosing the PC12 cells on the logarithmic growth phase, after cell count , inoculating thems in 96-well plates on the density of 104/mL. Culturing cells for 24 hours, exchange a half of nutrient medium,then add different concentrations of Aβ25-35(A:0μmol/L (control group)、B:10μmol/L、C:20μmol/L、D:30μmol/L、E:40μmol/L). Culturing cells for 24 hours, obtain the cell survival percentage,carry on the statistical analysis by the SPSS software, choosing variance analytic method. Discoverying the damage effect on PC-12 cells obvious when Aβ25-35 in the concentration of 30μmol/L. Culture the cells 24 hours according to the same condition and then add Aβ25-35 to them on the density of 30μmol/L, 12 hours later, add the gingko leaf extraction and the creatine phosphoric sodium according to the following grouping method : the Ginkgo biloba extract group A:Aβ0umol/L (control group), B:Aβ30umol/L,C:Aβ30umol/L + EGB 100mg/L , D: Aβ30u mol /L+ EGB200mg/L, E: Aβ30umol/L + EGB400mg/L); the creatine phosphate sodium group:(A: Aβ0umol/L (control group),B:Aβ30umol/L.C:Aβ30umol/L+PCr10mmol/L,D:Aβ30umol/L+PCr20mmol/L,E:Aβ30umol/L+PCr30mmol/L), culturing for 12 hours,and then use CCK-8 to measure the cell survival percentage,after statistical analysis, we draw a conclude: the Ginkgo biloba extract(200mg/L) and the creatine phosphate sodium group (20mmol/L) play obvious protective role in the damage. According to the above methods, we use Aβ25-35 (30μmol/L) to damage the PC12 cells, 12 hours later, add the Ginkgo biloba extract (200mg/L), the creatine phosphate sodium(20mmol/L) to protect cells,after raising 12 hours, mark the PC12 cell with line plastochondria membrane potential special fluorescent probe JC-1. Start the laser confocal microscopy, Choose the 488nm argon ion laser and the 543nm helium neon laser as the stimulation light, 530±15 nm and 615±30 nm channel as the reception channel. Use Lasersharp 2000 software to scan the picture, and carry on finally the image in the Laserpix software processing. Simultaneously choose the cell, in the concentration of 104/mL , to fling the piece at the speed of 600r/min, detect Apoptosis by TUNEL the reagent box of Apoptosis, Calculate the apoptosis rate, and collect the image,and then use SPSS statistical software for statistical analysis.
Results: When Aβ25-35 in the concentration of 30μmol/L, effects of injury to PC12 cells are obvious. It has significant differences from groups: 0μmol/L, 10μmol/L, 20μmol/L, (p<0.01) and similar to group of 40μmol/L (P>0.01). After intervention in different concentrations of Ginkgo biloba extract and sodium creatine phosphate,measure OD value,use the analysis of variance method to obtain that the group of EGB 200mg/L has significant differences with the group of Aβ30μmol/L and EGB 100mg/L (P<0.01) and has no significant differences with the group of EGB 400mg/L and Aβ0μmol/L (P>0.01) .The group of PCr 20mmol/L has obvious differences with the group of Aβ30μmol/L and PCr 10 mmol/L (P<0.01)and has no significant differences with the group of PCr 30 mmol/L and Aβ0μmol/L (P>0.01). Add Aβto PC12 cells in concentrations of 30umol/L(referred to as Group Aβ, the same below), after 24 hours, Mitochondrial membrane potential shows that red fluorescence intensity decreased significantly and green fluorescence intensity enhanced , which means that MMP lower. After dealing with PC12 cells according to the group of Aβ30umol/L+EGB 200mg/L (referred to as ginkgo group, the same below) and the group of Aβ30umol/L+ PCr20mmol / L (referred to as sodium creatine phosphate group, the same below), red fluorescence intensity markedly improved, and green fluorescence intensity decreased significantly, which means MMP increases. Group Aβ(30 umol/L Aβ), EGB (Aβ30 umol/L+EGB200mg/L) and PCr (Aβ30umol/L+PCr20mmol/L) have TUNEL-positive cells, that are, apoptotic cells,POD- marked green fluorescent cells significantly, DAB color,its nucleus is stained brown. The apoptosis rate of group Aβ(63%) is higher than that ginkgo group (21%) and creatine phosphate group (22%) (p <0.01).
Conclusion: 1.Aβ25-35 in the concentration of 30 umol/L can induce apoptosis in PC12 cells, establishing the cell type of Alzheimer’s disease. 2.Ginkgo biloba extract in the concen- tration of 200mg/L,creatine phosphate sodium in the concent- ration of 20 mmol/L can significantly increase the survival rate of cells by raising the level of mitochondrial membrane potential and the inhibition of apoptosis,and so play the role in protecting PC-12 cells injuried by Aβ25-35.
方法:取对数生长期PC12细胞,细胞计数后,以104/mL的密度接种于96孔板中,培养24小时后,半量换液,并加入不同浓度的Aβ25-35(A:0μmol/L (对照组)、B:10μmol/L、C:20μmol/L、D:30μmol/L、E:40μmol/L)。培养24小时后测得细胞存活率,应用SPSS软件选择方差分析法进行统计分析,发现在Aβ25-35浓度为30μmol/L时对PC12细胞的损伤效果明显。按相同条件进行细胞培养24小时后加入Aβ25-35,浓度为30μmol/L,12小时后,按下面分组方法分别加入银杏叶提取物及磷酸肌酸钠:银杏叶提取物A:Aβ0umol (对照组)、B:Aβ30umol/L、C:Aβ30umol/L +EGB100mg/L、D:Aβ30umol/L+EGB200mg/L、E:Aβ30umol /L +EGB400mg/L,磷酸肌酸钠组A:Aβ0umol/L (对照组)、B:Aβ30umol /L、C:Aβ30umol/L+PCr10mmol/L、D:Aβ30umol/L + Cr20mmol/L、E:Aβ30umol/L+PCr 30 mmol /L。培养12小时后,CCK-8法测细胞存活率,统计分析后得出,银杏叶提取物组200mg/L,磷酸肌酸钠组20mmol/L对细胞保护作用明显。按上述方法用Aβ25-35浓度为30μmol/L损伤PC12细胞,12小时后予银杏叶提取物组(200mg/L), PCr组(20mmol/L)保护细胞,培养12小时后,用线粒体膜电位特异荧光探针JC-1标记PC12细胞,启动共聚焦显微镜,选择488nm氩离子激光和543nm氦氖激光作为激发光,选择530±15 nm和615±30 nm通道作为接收通道,启动计算机运行Lasersharp2000软件,扫描图象,最后将图象在Laserpix软件中进行处理。同时将浓度为104/mL的细胞,以600转/分的速度进行细胞甩片后,按TUNEL凋亡试剂盒的操作步骤检测细胞凋亡,计算细胞凋亡率,采集图像。采用SPSS统计软件进行统计学分析。
结果:在Aβ25-35浓度为30μmol/L时,对PC12细胞损伤效果明显,与0μmol/L,10μmol/L ,20μmol/L组有显著差异( P < 0.01 ),而和40μmol/L组,差异不明显(P> 0.01)。以不同浓度的银杏叶提取物及磷酸肌酸钠进行干预后,测量OD值,采用方差分析法得出EGB200mg/L组与Aβ30umol /L,EGB 100 mg/L组差异明显(P<0.01),而和400 mg/L组及Aβ0 umol /L组无明显差异(P>0.01),PCr 20 mmol/L与Aβ30umol /L,PCr 10 mmol/L组差异明显(P< 0.01 ),和PCr 30mmol/L组无明显差异。线粒体膜电位示30umol/L的Aβ处理PC12细胞(简称为Aβ组,下同)24小时后,红色荧光强度明显降低,绿色荧光强度明显增强,即线粒体膜电位(MMP)降低。而以Aβ30umol/L + EGB200mg/L组处理PC12细胞(简称为银杏组,下同)及Aβ30umol/L+PCr 20mmol/L组同时处理PC12细胞(简称为磷酸肌酸钠组,下同)后,红色荧光强度明显增强,绿色荧光强度明显降低,即线粒体膜电位(MMP)升高。Aβ组(30umol/L Aβ)、EGB组(Aβ30 umol/L + EGB 200mg/L)及PCr组(Aβ30umol/L + PCr 20mmol/L)均可见TUNEL阳性细胞,即凋亡细胞,PO-D标记的凋亡细胞显绿色荧光;而以DAB显色,凋亡细胞核被染成棕色。Aβ组细胞凋亡率(63%)高于银杏组(21%)及磷酸肌酸组(22%)(p<0.01)。
结论:1.浓度为30umol/L的Aβ25-35可诱导PC12细胞凋亡,建立AD的细胞膜型。2.浓度为200mg/L的银杏叶提取物及20mmol/L的磷酸肌酸钠,通过提高PC12细胞的线粒体膜电位水平,抑制细胞凋亡,提高细胞存活率,从而对Aβ25-35损伤的PC12细胞起到一定的保护作用。
Objective: This experiment uses amyloidβ(Aβ) 25-35 to induce apoptosis in PC12 cells, establishing the cell type of Alzheimer’s disease, and then provides Ginkgo biloba extract and creatine phosphate sodium to intervene. To explore whether the two play protective roles in neuronal apoptosis induced by Aβ25-35.
Materials and methods: Choosing the PC12 cells on the logarithmic growth phase, after cell count , inoculating thems in 96-well plates on the density of 104/mL. Culturing cells for 24 hours, exchange a half of nutrient medium,then add different concentrations of Aβ25-35(A:0μmol/L (control group)、B:10μmol/L、C:20μmol/L、D:30μmol/L、E:40μmol/L). Culturing cells for 24 hours, obtain the cell survival percentage,carry on the statistical analysis by the SPSS software, choosing variance analytic method. Discoverying the damage effect on PC-12 cells obvious when Aβ25-35 in the concentration of 30μmol/L. Culture the cells 24 hours according to the same condition and then add Aβ25-35 to them on the density of 30μmol/L, 12 hours later, add the gingko leaf extraction and the creatine phosphoric sodium according to the following grouping method : the Ginkgo biloba extract group A:Aβ0umol/L (control group), B:Aβ30umol/L,C:Aβ30umol/L + EGB 100mg/L , D: Aβ30u mol /L+ EGB200mg/L, E: Aβ30umol/L + EGB400mg/L); the creatine phosphate sodium group:(A: Aβ0umol/L (control group),B:Aβ30umol/L.C:Aβ30umol/L+PCr10mmol/L,D:Aβ30umol/L+PCr20mmol/L,E:Aβ30umol/L+PCr30mmol/L), culturing for 12 hours,and then use CCK-8 to measure the cell survival percentage,after statistical analysis, we draw a conclude: the Ginkgo biloba extract(200mg/L) and the creatine phosphate sodium group (20mmol/L) play obvious protective role in the damage. According to the above methods, we use Aβ25-35 (30μmol/L) to damage the PC12 cells, 12 hours later, add the Ginkgo biloba extract (200mg/L), the creatine phosphate sodium(20mmol/L) to protect cells,after raising 12 hours, mark the PC12 cell with line plastochondria membrane potential special fluorescent probe JC-1. Start the laser confocal microscopy, Choose the 488nm argon ion laser and the 543nm helium neon laser as the stimulation light, 530±15 nm and 615±30 nm channel as the reception channel. Use Lasersharp 2000 software to scan the picture, and carry on finally the image in the Laserpix software processing. Simultaneously choose the cell, in the concentration of 104/mL , to fling the piece at the speed of 600r/min, detect Apoptosis by TUNEL the reagent box of Apoptosis, Calculate the apoptosis rate, and collect the image,and then use SPSS statistical software for statistical analysis.
Results: When Aβ25-35 in the concentration of 30μmol/L, effects of injury to PC12 cells are obvious. It has significant differences from groups: 0μmol/L, 10μmol/L, 20μmol/L, (p<0.01) and similar to group of 40μmol/L (P>0.01). After intervention in different concentrations of Ginkgo biloba extract and sodium creatine phosphate,measure OD value,use the analysis of variance method to obtain that the group of EGB 200mg/L has significant differences with the group of Aβ30μmol/L and EGB 100mg/L (P<0.01) and has no significant differences with the group of EGB 400mg/L and Aβ0μmol/L (P>0.01) .The group of PCr 20mmol/L has obvious differences with the group of Aβ30μmol/L and PCr 10 mmol/L (P<0.01)and has no significant differences with the group of PCr 30 mmol/L and Aβ0μmol/L (P>0.01). Add Aβto PC12 cells in concentrations of 30umol/L(referred to as Group Aβ, the same below), after 24 hours, Mitochondrial membrane potential shows that red fluorescence intensity decreased significantly and green fluorescence intensity enhanced , which means that MMP lower. After dealing with PC12 cells according to the group of Aβ30umol/L+EGB 200mg/L (referred to as ginkgo group, the same below) and the group of Aβ30umol/L+ PCr20mmol / L (referred to as sodium creatine phosphate group, the same below), red fluorescence intensity markedly improved, and green fluorescence intensity decreased significantly, which means MMP increases. Group Aβ(30 umol/L Aβ), EGB (Aβ30 umol/L+EGB200mg/L) and PCr (Aβ30umol/L+PCr20mmol/L) have TUNEL-positive cells, that are, apoptotic cells,POD- marked green fluorescent cells significantly, DAB color,its nucleus is stained brown. The apoptosis rate of group Aβ(63%) is higher than that ginkgo group (21%) and creatine phosphate group (22%) (p <0.01).
Conclusion: 1.Aβ25-35 in the concentration of 30 umol/L can induce apoptosis in PC12 cells, establishing the cell type of Alzheimer’s disease. 2.Ginkgo biloba extract in the concen- tration of 200mg/L,creatine phosphate sodium in the concent- ration of 20 mmol/L can significantly increase the survival rate of cells by raising the level of mitochondrial membrane potential and the inhibition of apoptosis,and so play the role in protecting PC-12 cells injuried by Aβ25-35.
引文
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29 赵振军,张丽杰,易小兵.几种凋亡细胞检测方法的比较. 医学综述,2003,9 (7):387~388
30 陈罡,王晓冬. 细胞凋亡检测技术的进展. 国外医学分子生物学分册,2003,25(2):125~128
31 杨国锋,姚建华,彭立威.银杏叶提取物对海马注射淀粉β样蛋白致阿尔茨海默病大鼠模型神经元凋亡的保护作用. 中国临床康复,2006,10(11):42~44
32 Zimmermann M,Colciaghi F,Cattabeni F,et al.Ginkgo biloba extract from molecular mechanisms to the treatment of Alzhelmer's disease.Cell Mol Biol(Noisyle-gran),2002,48(6):613~623
33 肖淑萍.银杏叶提取物对经铝化物诱导的阿尔茨海默病模型大鼠的影响. 中国基层医药,2006,13(3):405 ~408
34 秦历杰 .外源性磷酸肌酸治疗急性脑梗死的疗效观察. 中国实用神经疾病杂志,2007,10(3):31~32
35 曹倩,邢泉生,李自普.磷酸肌酸停搏液对法洛四联症患儿的心肌保护作用. 中国体外循环杂,2007,5(1):14~16
36 侯立向.一种心肌保护剂:磷酸肌酸. 生物化学与生物物理进展,2003,30 (2):324
37 Ikeda K, Iwasaki Y, Kinoshita M.Oral administration of creatine monohydrate retards progression of motor neuron disease in the wobbler mouse. Amyotroph Lateral Scler Other Motor Neuron Disord,2000, 1(3):207 ~ 212
38 Matthews RT,Yang L,Jenkins BG,et al.Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington’s disease. J. Neurosci,1998,18:156~ 163
39 Matthews RT, Ferrante RJ, Klivenyi P, et al.Creatine and cyclocreatine attenuate MPTP neurotoxicity. Exp Neurol, 1999,157(1):142~149
40 Xu CJ , Klunk WE, Kanfer JN, et al.Phosphocreatine-depen dent glutamate uptake by synaptic vesicles. A comparison with atp-dependent glutamate uptake. J. Biol. Chem, 1996 , 271 (23):13435~13440
41 Brustovetsky N, Brustovetsky T, Dubinsky JM.On the mechanisms of neuroprotection by creatine and phosphocr eatine. J. Neurochem, 2001,76:425~434
42 Wallimann T, Wyss M,Brdiczka D, et al. Intracellularcompartmentation,structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands:the 'phosphocreatine circuit' for cellular energy homeostasis[J]. Biochemistry Journal, 1992, 281: 21 ~40
43 Hemmer W, Wallimann T.Functional aspects of creatine kinase in brain. Dev Neurosci, 1993,15(3-5):249 ~260
44 Kinnally KW, Lohret TA, Campo ML, Mannella CA. Perspectives on the mitochondrial multiple conductance channel. J Bioenerg Biomembr,1996,28(2):115~123
45 O'Gorman E, Beutner G, Dolder M.The role of creatine kinase inhibition of mitochondrial permeability transition. FEBS Lett, 1997,414(2), 253~ 257
1 Jeyashekan NS, Sandan A, Vo-dinht T. Protein amyloidose misfolding : mechanisms, detection , and pathological imp- lications. Methods Mol Biol, 2005, 300: 417~435
2 Engidawork E, Gulesserian T, Yoo BC, et al. Alteration of caspases and apoptosis-related proteins in brains of patients with Alzheimer's disease. Biochem Biophys Res Commun , 2001, 281(1):84~93
3 Luo X, Weber GA, Zheng J, et al. C1q-calreticul ininduced oxidative neurotoxicity: relevance for the neuropathogenesi -s of Alzheimer’s disease . J Neuroimmunol, 2003, 135 (1 - 2) :62~71
4 姜招峰.β淀粉样蛋白、氧自由基和腺苷及其受体的相互作用. 国外医学·老年医学分册,2002,23(2):57~59
5 Reyes AE, Chacon MA, Dinamarca MC, et al. Acetylcholi- nesterase–Abeta complexes are more toxic than Abeta fibrils in rat hippocampus: effect on rat beta-amyloid aggregation, laminin expression, reactive as astrocytosis, and neuronal cell loss. Am J Pathol, 2004,164(6):2163 ~2174
6 Qiu Z, Gruol DL.Interleukin-6, beta-amyloid peptide and NMDA interactions in rat cortical neurons. J Neuroim- munol, 2003 ,139(1-2):51~57
7 Rberds SL, Anderson J, Basi G,et al. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer’s diseasetherapeutics. Hum Mol Genet,2001,10(1):1317~ 1324
8 Stephan A, Laroche S, Davis S. Learning deficits and dysfunctional synaptic plasticity induced by aggregated amyloid deposits in the dentate gyrus are rescued by chronic treatment with indomethacin. Eur J Neurosci, 2003, 17 (9) : 1921~1927
9 Prasad KN, La Rosa FG, Prasad JE.Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and beta-amyloid. In Vitro Cell Dev Biol Anim,1998,34(3):265~274
10 Parvathy S, Rajadas J, Ryan H, et al.Abeta peptide conformation determines uptake and interleukin-1alpha expression by primary microglial cells.Neurobiol Aging, 2008 Mar 11 [Epub ahead of print]
11 Bhatia R, Lin H, Lal R. Fresh and globular amyloid beta protein (1-42) induces rapid cellular degeneration:evidence for Abeta-P channel-mediated cellular toxicity. FASEB J, 2000, 14(9):1233~1243
12 Micelli S, Meleleo D, Picciarelli V, et al. Effect of sterols on beta-amyloid peptide (AbetaP1- 40) channel formation and their properties in planar lipid membranes. Biophys J, 2004,86:2231~2237
13 Fang Ba, Peter KT, Christina G. The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl- 4- phenyl- 1, 2, 3, 6- tetrahydropyridine (MPTP) cytotoxic models. NeurochemInt, 2004, 45(1):31~38
14 Bobich JA, Zheng Q,Campbell A.Incubation of nerve endings with a physiological concentration of Abeta1-42 activates CaV2.2(N-Type)-voltage operated calcium channels and acutely increases glutamate and noradrenaline release. J Alzheimers Dis,2004 ,6(3) :243 ~255
15 MacManus A, Ramsden M, Murray M ,et al. Enhancement of (45)Ca(2+) influx and voltage-dependent Ca(2+) channel activity by beta–amyloid-(1-40) in rat cortical synapto- somes and cultured cortical neurons. Modulation by the proinflammatory cytokine interleukin-1beta. J Biol Chem, 2000, 275(7):4713~ 4718
16 任琳戴,雪伶,姜招峰.金属离子与阿尔茨海默病的关系.生命的化学,2007,27 (1 ):64~67
17 Tanzi RE. The synaptic Abeta hypothesis of Alzheimer disease . NatNeurosci, 2005, 8:977 ~979
18 Kadir A,Andreasen N, Almkvist O, et al. Effect of phenserine treatment on brain functional activity and amyloid in Alzheimer's disease. Ann Neurol, 2008, 2 ( 25) [Epub ahead of print]
19 Birks J. The evidence for the efficacy of cholinesterase inhibitors in the treatment of Alzheimer's disease is convincing . Int Psychogeriatr, 2008,20(2):279~286
20 Little JT, Walsh S, Aisen PS. An update on huperzine A as a treatment for Alzheimer's disease. Expert Opin Investig. Drugs, 2008 ,17(2):209~215
21 Okura Y, MatsumotoY. Development of anti-Abeta vaccination as a promising therapy for Alzheimer's disease. .Drug News Perspect, 2007,20(6):379~386
22 Austen BM, Paleologou KE, Ali SA, et al.Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide. Biochemistry, 2008, 47(7):1984 ~1992
23 杨斯柳,顾卫红. 神经变性病与抗氧化剂. 中日友好医院学报,2007,21(4):235~237
24 陈晓红, 王荫华, 阿尔茨海默病的治疗新进展. 中国全科医学,2001 , 4(12): 940~942
25 Peacock JM , Folsom AR , Knopman DS , et al Association of nonsteroidal antiinflammatory drugs and aspirin with cognitive performance in middle aged adults Neuro epidemiology , 1999 ,18 (3) : 134~143
26 Tobinick EL, Gross H. Rapid cognitive improvement in Alzheimer's disease following perispinal etanercept adminis- tration . Neuroinflammation, 2008, 9(5):2
27 Price KA, Crouch PJ, White AR. Therapeutic treatment of Alzheimer's disease using metal complexing agents. .Recent Patents CNS Drug Discov, 2007,2(3):180~187
28 Yagami T , Ueda K, Sakaeda T , et al1. Protective effects of a selective L-type vollage-sensitive calcium channel blocker , S-312-d,on neuronal cell death . Biochemical Pharma- cology ,2004,67(6): 1153~1165
29 罗京京,何玲,刘国卿.治疗阿尔茨海默新药盐酸美金刚.中国新药杂志,2006,15(9):742~744
30 Glodzik L, King KG, Gonen O,et al.Memantine decreases hippocampal glutamate levels:A magnetic resonance spectro -scopy study. Prog Neuropsychopharmacol Biol Psychiatry, 2008,Feb 7 [Epub ahead of print]
31 邹士宗.阿尔茨海默病的药物治疗. 中国煤炭工业医学杂志,2007,10(5):497~498
2 叶伟,唐孝威.老年性痴呆症发病机制及其防治措施的研究进展. 国外医学遗传学分册,2005,28 (6):379 ~ 381
3 Jie K, Hans - Geong L , Unterbeek A , et al . The Precursor of Alzheimer disease amyloid A4 protein resembles a cell -surface receptor. Nature ,1987,325 (19) : 733 ~735
4 Verdile G, Fuller S, Atwood CS, et al. The role of beta amyloid in Alzheimer's disease : still a cause of everything or the only one who got caught .Pharmacol Res., 2004, 50 (4):397~409
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6 Joanne M, Rirka G, Anna J, et al. Inositol steroisomer Stebilize an oligometric aggregate of Alzhemier abeta– induced toxicity. J Biol Chem ,2000 ,275(24):18495 ~18502
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8 Scripnikov A, Khomenko A, Napryeyenko O,et al. Effects of Ginkgo biloba extract EGb 761 on neuropsychiatric symptoms of dementia : findings from a randomised controlled trial. Wien Med Wochenschr,2007,157(13- 14):295~300
9 Nickolay Brustovetsky, Tatiana Brustovetsky,Janet M.et al. On the mechanisms of neuroprotection by creatine and phosphocreatine. Journal of Neurochemistry,2001,76:425 ~434
10 ON Hausmann, K Fouad, T Wallimann,et al. Protective effects of oral creatine supplementation on spinal cord injury in rats. Spinal Cord ,2002,40:449 ~ 456
11 Peter Klivenyi, Mahmoud Kiaei, Gabrielle Gardian,et al. Additive neuroprotective effects of creatine and cyclo- oxygenase - inhibitors in a transgenic mouse model of amyotrophic lateral sclerosis. Journal of Neurochemistry, 2004, 88: 576~582
12 房绍宽,吴江,周春奎.褪黑素对 Aβ25-35 诱发 PC12 细胞损伤的保护作用. 中国老年学杂志,2007,27:3~5
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14 McLaurin J, Golomb R, Jurewicz A , et al. Inositol steroisomer stebilize an oligometric aggregate of Alzhemier amyloid beta peptide and inhibit abeta-induced toxicity. J Biol Chem ,2000 ,275(24) : 18495~ 18502
15 Sic L , Michael D , Clark P , et al . Presenillin-1 mutations increase levels of ryanodien receptors and calcium release in PC12 cell and cortical neurons. J Biol Chem, 2000, 275 (24) :19195~ 19200
16 王亚利,宋天保,路明.Aβ诱导 PC-12 细胞凋亡建立阿尔茨海默病细胞模型. 西安医科大学学报,2002,2 (4) :129~ 132
17 Luo X, Weber GA, Zheng J, et al. C1q-calreticulin induced oxidative neurotoxicity:relevance for the neuropathogenesis of Alzheimer’s disease. J Neuroimmunol,2003,135 ( 1-2) : 62~71
18 Reyes AE,Chacon MA,Dinamarca MC,et al.Acetylcholin- esterase-Abeta complexes are more toxic than Abeta fibrils in rat hippocampus:effect on rat beta-amyloid aggregation, laminin expression,reactive astrocytosis,and neuronal cell loss. Am J Pathol,2004;164(6):2163 ~2174
19 Rberds SL,Anderson J,Basi G,et al.BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain:implications for Alzheimer’s disease therapeutics. Hum Mol Genet,2001,10 (1):1317~1324
20 Bhatia R,Lin H,Lal R.Fresh and globular amyloid beta protein(1-42) induces rapid cellular degeneration:evidence for AbetaP channel-mediated cellular toxicity. FASEB J, 2000, 14: 1233~1243
21 Micelli S, Meleleo D,Picciarelli V,et al. Effect of sterols on beta-amyloid peptide (AbetaP1-40) channel formation and their properties in planar lipid membranes. Biophys J, 2004,86:2231~2237
22 王铁枫, 贺娟.阿尔茨海默病发病机制研究进展.中国行为医学科学,2003,12 (4) :476~478
23 林娜. 线粒体与细胞凋亡. 国外医学遗传学册,2000,23(2) :89~91
24 Granville DJ, Gottlieb RA. Mitochondria as regulators of cell death and survival. ScientificWorldJournal,2002,2 : 1569~1578
25 Ly JD,Grubb DR,Lawen A.The mitochondrial membrane potential(deltapsi(m)) in apoptosis:an update[J]. Apoptosis, 2003,8(2):115~128
26 Hirsch T,Marzo I,Kroemer G.Role of the mitochondrial permeability transition pore in apoptosis. Biosci Rep, 1997, 17 (1) :67~76
27 王嘉宁,郭宁.细胞凋亡的检测技术与方法. 中国药理学与毒理学杂志,2005,19(6):466~470
28 赵利红,崔利德,李陈莉.细胞凋亡的检测方法. 河北医药,2005,27(10):783~785
29 赵振军,张丽杰,易小兵.几种凋亡细胞检测方法的比较. 医学综述,2003,9 (7):387~388
30 陈罡,王晓冬. 细胞凋亡检测技术的进展. 国外医学分子生物学分册,2003,25(2):125~128
31 杨国锋,姚建华,彭立威.银杏叶提取物对海马注射淀粉β样蛋白致阿尔茨海默病大鼠模型神经元凋亡的保护作用. 中国临床康复,2006,10(11):42~44
32 Zimmermann M,Colciaghi F,Cattabeni F,et al.Ginkgo biloba extract from molecular mechanisms to the treatment of Alzhelmer's disease.Cell Mol Biol(Noisyle-gran),2002,48(6):613~623
33 肖淑萍.银杏叶提取物对经铝化物诱导的阿尔茨海默病模型大鼠的影响. 中国基层医药,2006,13(3):405 ~408
34 秦历杰 .外源性磷酸肌酸治疗急性脑梗死的疗效观察. 中国实用神经疾病杂志,2007,10(3):31~32
35 曹倩,邢泉生,李自普.磷酸肌酸停搏液对法洛四联症患儿的心肌保护作用. 中国体外循环杂,2007,5(1):14~16
36 侯立向.一种心肌保护剂:磷酸肌酸. 生物化学与生物物理进展,2003,30 (2):324
37 Ikeda K, Iwasaki Y, Kinoshita M.Oral administration of creatine monohydrate retards progression of motor neuron disease in the wobbler mouse. Amyotroph Lateral Scler Other Motor Neuron Disord,2000, 1(3):207 ~ 212
38 Matthews RT,Yang L,Jenkins BG,et al.Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington’s disease. J. Neurosci,1998,18:156~ 163
39 Matthews RT, Ferrante RJ, Klivenyi P, et al.Creatine and cyclocreatine attenuate MPTP neurotoxicity. Exp Neurol, 1999,157(1):142~149
40 Xu CJ , Klunk WE, Kanfer JN, et al.Phosphocreatine-depen dent glutamate uptake by synaptic vesicles. A comparison with atp-dependent glutamate uptake. J. Biol. Chem, 1996 , 271 (23):13435~13440
41 Brustovetsky N, Brustovetsky T, Dubinsky JM.On the mechanisms of neuroprotection by creatine and phosphocr eatine. J. Neurochem, 2001,76:425~434
42 Wallimann T, Wyss M,Brdiczka D, et al. Intracellularcompartmentation,structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands:the 'phosphocreatine circuit' for cellular energy homeostasis[J]. Biochemistry Journal, 1992, 281: 21 ~40
43 Hemmer W, Wallimann T.Functional aspects of creatine kinase in brain. Dev Neurosci, 1993,15(3-5):249 ~260
44 Kinnally KW, Lohret TA, Campo ML, Mannella CA. Perspectives on the mitochondrial multiple conductance channel. J Bioenerg Biomembr,1996,28(2):115~123
45 O'Gorman E, Beutner G, Dolder M.The role of creatine kinase inhibition of mitochondrial permeability transition. FEBS Lett, 1997,414(2), 253~ 257
1 Jeyashekan NS, Sandan A, Vo-dinht T. Protein amyloidose misfolding : mechanisms, detection , and pathological imp- lications. Methods Mol Biol, 2005, 300: 417~435
2 Engidawork E, Gulesserian T, Yoo BC, et al. Alteration of caspases and apoptosis-related proteins in brains of patients with Alzheimer's disease. Biochem Biophys Res Commun , 2001, 281(1):84~93
3 Luo X, Weber GA, Zheng J, et al. C1q-calreticul ininduced oxidative neurotoxicity: relevance for the neuropathogenesi -s of Alzheimer’s disease . J Neuroimmunol, 2003, 135 (1 - 2) :62~71
4 姜招峰.β淀粉样蛋白、氧自由基和腺苷及其受体的相互作用. 国外医学·老年医学分册,2002,23(2):57~59
5 Reyes AE, Chacon MA, Dinamarca MC, et al. Acetylcholi- nesterase–Abeta complexes are more toxic than Abeta fibrils in rat hippocampus: effect on rat beta-amyloid aggregation, laminin expression, reactive as astrocytosis, and neuronal cell loss. Am J Pathol, 2004,164(6):2163 ~2174
6 Qiu Z, Gruol DL.Interleukin-6, beta-amyloid peptide and NMDA interactions in rat cortical neurons. J Neuroim- munol, 2003 ,139(1-2):51~57
7 Rberds SL, Anderson J, Basi G,et al. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer’s diseasetherapeutics. Hum Mol Genet,2001,10(1):1317~ 1324
8 Stephan A, Laroche S, Davis S. Learning deficits and dysfunctional synaptic plasticity induced by aggregated amyloid deposits in the dentate gyrus are rescued by chronic treatment with indomethacin. Eur J Neurosci, 2003, 17 (9) : 1921~1927
9 Prasad KN, La Rosa FG, Prasad JE.Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and beta-amyloid. In Vitro Cell Dev Biol Anim,1998,34(3):265~274
10 Parvathy S, Rajadas J, Ryan H, et al.Abeta peptide conformation determines uptake and interleukin-1alpha expression by primary microglial cells.Neurobiol Aging, 2008 Mar 11 [Epub ahead of print]
11 Bhatia R, Lin H, Lal R. Fresh and globular amyloid beta protein (1-42) induces rapid cellular degeneration:evidence for Abeta-P channel-mediated cellular toxicity. FASEB J, 2000, 14(9):1233~1243
12 Micelli S, Meleleo D, Picciarelli V, et al. Effect of sterols on beta-amyloid peptide (AbetaP1- 40) channel formation and their properties in planar lipid membranes. Biophys J, 2004,86:2231~2237
13 Fang Ba, Peter KT, Christina G. The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl- 4- phenyl- 1, 2, 3, 6- tetrahydropyridine (MPTP) cytotoxic models. NeurochemInt, 2004, 45(1):31~38
14 Bobich JA, Zheng Q,Campbell A.Incubation of nerve endings with a physiological concentration of Abeta1-42 activates CaV2.2(N-Type)-voltage operated calcium channels and acutely increases glutamate and noradrenaline release. J Alzheimers Dis,2004 ,6(3) :243 ~255
15 MacManus A, Ramsden M, Murray M ,et al. Enhancement of (45)Ca(2+) influx and voltage-dependent Ca(2+) channel activity by beta–amyloid-(1-40) in rat cortical synapto- somes and cultured cortical neurons. Modulation by the proinflammatory cytokine interleukin-1beta. J Biol Chem, 2000, 275(7):4713~ 4718
16 任琳戴,雪伶,姜招峰.金属离子与阿尔茨海默病的关系.生命的化学,2007,27 (1 ):64~67
17 Tanzi RE. The synaptic Abeta hypothesis of Alzheimer disease . NatNeurosci, 2005, 8:977 ~979
18 Kadir A,Andreasen N, Almkvist O, et al. Effect of phenserine treatment on brain functional activity and amyloid in Alzheimer's disease. Ann Neurol, 2008, 2 ( 25) [Epub ahead of print]
19 Birks J. The evidence for the efficacy of cholinesterase inhibitors in the treatment of Alzheimer's disease is convincing . Int Psychogeriatr, 2008,20(2):279~286
20 Little JT, Walsh S, Aisen PS. An update on huperzine A as a treatment for Alzheimer's disease. Expert Opin Investig. Drugs, 2008 ,17(2):209~215
21 Okura Y, MatsumotoY. Development of anti-Abeta vaccination as a promising therapy for Alzheimer's disease. .Drug News Perspect, 2007,20(6):379~386
22 Austen BM, Paleologou KE, Ali SA, et al.Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide. Biochemistry, 2008, 47(7):1984 ~1992
23 杨斯柳,顾卫红. 神经变性病与抗氧化剂. 中日友好医院学报,2007,21(4):235~237
24 陈晓红, 王荫华, 阿尔茨海默病的治疗新进展. 中国全科医学,2001 , 4(12): 940~942
25 Peacock JM , Folsom AR , Knopman DS , et al Association of nonsteroidal antiinflammatory drugs and aspirin with cognitive performance in middle aged adults Neuro epidemiology , 1999 ,18 (3) : 134~143
26 Tobinick EL, Gross H. Rapid cognitive improvement in Alzheimer's disease following perispinal etanercept adminis- tration . Neuroinflammation, 2008, 9(5):2
27 Price KA, Crouch PJ, White AR. Therapeutic treatment of Alzheimer's disease using metal complexing agents. .Recent Patents CNS Drug Discov, 2007,2(3):180~187
28 Yagami T , Ueda K, Sakaeda T , et al1. Protective effects of a selective L-type vollage-sensitive calcium channel blocker , S-312-d,on neuronal cell death . Biochemical Pharma- cology ,2004,67(6): 1153~1165
29 罗京京,何玲,刘国卿.治疗阿尔茨海默新药盐酸美金刚.中国新药杂志,2006,15(9):742~744
30 Glodzik L, King KG, Gonen O,et al.Memantine decreases hippocampal glutamate levels:A magnetic resonance spectro -scopy study. Prog Neuropsychopharmacol Biol Psychiatry, 2008,Feb 7 [Epub ahead of print]
31 邹士宗.阿尔茨海默病的药物治疗. 中国煤炭工业医学杂志,2007,10(5):497~498