【作者】 刘希奇；

【导师】 张国华； 杨国锋；

【作者基本信息】 河北医科大学 ， 神经病学， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原