Aβ_1-40诱导的神经干细胞凋亡中钾通道和JNK信号转导通路的变化

【作者】 田嘉莹；

【导师】 盛宝英； 齐志国；

【作者基本信息】 佳木斯大学 ， 神经病学（专业学位）， 2013， 硕士

【摘要】 目的：探讨钾通道在Aβl-40诱导的神经干细胞凋亡过程中发挥的作用；探讨JNK信号转导通路在Aβl-40诱导的神经干细胞凋亡中的作用；以及钾通道与JNK的关系。进一步了解阿尔茨海默病（Alzheimer’s disease, AD）的发病机制，从而为开发治疗AD药物提供新的思路和方法。方法：1.分离提取新生的Wistar大鼠海马区的神经干细胞并传代培养，应用Nestin细胞免疫化学法鉴定。2.应用MTT法检测TEA干预Aβ_1-40诱导后的神经干细胞存活率的变化。3.应用Hoechst33342染色法在荧光显微镜紫外激发下观察神经干细胞凋亡的形态学变化。4.应用比色法检测TEA对Aβ_1-40诱导的神经干细胞Caspase-3活性的变化。5.应用Western blot法检测Aβ_1-40诱导神经干细胞不同时间点JNK磷酸化的表达。6.应用MTT法检测SP600125干预Aβl-40诱导的神经干细胞存活率的变化。7.应用Hoechst33342染色法在荧光显微镜紫外激发下观察神经干细胞凋亡的形态学变化。8.应用比色法检测SP600125干预后Aβ_1-40诱导的神经干细胞Caspase-3活性的变化。9.由于Aβl-40诱导的神经干细胞JNK磷酸化激活在24h达到高峰，故在这个时间点，孵育前30min加入TEA5mM，应用Western blot方法检测TEA对Aβl-40诱导的神经干细胞JNK磷酸化的影响。结果：1.12h内新生大鼠海马区分离的单个神经干细胞相互聚集成团状，3d后呈透亮的圆形，传代培养至第三代可见培养液中形成较大的神经球，神经干细胞标记性蛋白Nestin的表达阳性。2.1TEA对Aβ_1-40诱导的神经干细胞存活率的影响，结果显示：与空白对照组相比Aβ_1-40组神经干细胞存活数明显减少（p＜0.05），且随时间延长神经干细胞存活率下降。与Aβ_1-40组相比Aβ_1-40+TEA组神经干细胞存活率明显升高（p＜0.05）。2.2TEA对Aβ_1-40诱导的神经干细胞凋亡的影响。结果显示：荧光显微镜下Aβl-40组神经干细胞48h后出现部分细胞核呈浓染的碎块状，为典型的凋亡形态学改变。与Aβl-40组相比Aβ_1-40＋TEA组神经干细胞凋亡形态学变化少，凋亡率11.6%±0.4%，差异具有显著性（p＜0.01）。2.3TEA对Aβ_1-40诱导的神经干细胞Caspase-3活性的影响。结果显示：与Aβl-40组相比Aβ_1-40＋TEA组在各时间点均可降低Caspase-3的表达（p＜0.05），特别在24h这个时间点更为明显（p＜0.01），TEA可明显阻止Aβ_1-40诱导凋亡蛋白Caspase-3的发生。3.1Aβ_1-40诱导神经干细胞JNK磷酸化的表达。结果显示：在不同时间点，神经干细胞经Aβ_1-405μM孵育后，JNK的磷酸化均有表达。与空白对照组相比Aβ_1-40组JNK磷酸化水平随着时间的延长表达逐渐增加，在24h达到高峰，（p＜0.01）。3.2SP600125对Aβ_1-40诱导的神经干细胞存活率的影响。结果显示：与Aβ_1-40组相比Aβl-40＋SP600125组可明显减轻神经干细胞凋亡，且随时间的延长神经干细胞存活率逐渐升高。3.3SP600125对Aβ_1-40诱导的神经干细胞凋亡的影响。结果显示：与Aβ_1-40组相比Aβ_1-40＋SP600125组可明显降低神经干细胞凋亡的发生，Aβ_1-40＋SP600125组使凋亡发生率从20.3%±0.6%降至9.6%±1.3%（p＜0.01），而SP600125组无明显变化。3.4SP600125对Aβ_1-40诱导的神经干细胞Caspase-3活性的影响。结果显示：与Aβ_1-40组相比Aβ_1-40＋SP600125组随着时间的延长可显著减少Caspase-3的激活；Aβ_1-40组与Aβ_1-40＋SP600125组相比，差异具有显著性（p＜0.01）。4.TEA对Aβ_1-40诱导的神经干细胞JNK磷酸化表达的影响。结果显示：与Aβ_1-40组相比Aβ_1-40＋TEA组JNK磷酸化表达随时间的延长显著降低（p＜0.01）。而空白对照组和TEA组神经干细胞JNK磷酸化表达无明显改变，（p＞0.05）。结论：1.钾通道阻滞剂TEA可以明显减轻Aβ_1-40诱导的神经干细胞凋亡，使神经干细胞的存活率升高，凋亡率下降。2.Aβ_1-40可诱导神经干细胞发生JNK磷酸化，JNK信号转导通路参与了凋亡信号的转导，说明JNK在Aβ_1-40诱导神经干细胞损伤中具有重要的作用。3.JNK特异性阻断剂SP600125可抑制Aβ_1-40诱导的神经干细胞凋亡，对Aβ_1-40诱导的神经干细胞毒性具有明显的保护作用。更多还原

【Abstract】 Objective: Investigate the role of potassium channels in Aβ_1-40induced Neural stem cellsthe process of apoptosis; Explore JNK signal transduction pathway play a role in Aβ_1-40inducedthe process of apoptotic Neural stem cells; And the relationship between potassium channel andJNK. To further understand the pathogenesis of alzheimer’s disease, which offers a new ideasand method for treating Alzheimer’s disease to develop medicines.Methods:1To separate and extract nascent Wistar rats’s Neural stem cells of hippo-campus area, Then they were subcultured. passing on the three generations after gaining stableNeural stem cells which were detected thought Nestin of immunocytochemistry method.2Previously added TEA5mM,30minutes later, the survival rate of Neural stem cells at differenttime points（0h、12h、24h、48h）by MTT assay. TEA intervene in Aβ_1-40-induced Neuralstem cells, Then record the change of the Neural stem cell survival rate.3Previously addedTEA5mM,30minutes later, Neural stem cells are chronic incubated with Aβ_1-405μm,observed morphological changes of Neural stem cell under a fluorescence microscope,apoptosis was detected by Hoechst33342staining method.4Previously added TEA5mM,30minutes later, Neural stem cells are chronic incubated with Aβ_1-405μm at each point in time（0h、12h、24h、48h）, Expression of Caspase-3specific activity calculated by colorimetricmethod. To detect the effects of TEA act on Aβ_1-40-induced Neural stem cells.5To examine theexpression of Aβ_1-40-induced Neural stem cells JNK phosphorylation by Western blot. Torecord the expression of JNK phosphorylation in Neural stem cells by Aβ_1-405μM incubated fordifferent time（0h、12h、24h、48h）.6Incubated30min before adding SP60012510μΜ,Aβ_1-405μM and Neural stem cells were incubated at each time point （0h、12h、24h、48h）,Detected SP60012510μM act on Aβ_1-40-induced Neural stem cells survival rate by MTTmethod.7Previously added SP60012510μM,30minutes later, Neural stem cells are chronicincubated with Aβ_1-405μΜ, observed morphological changes of Neural stem cell under afluorescence microscope, apoptosis was detected by Hoechst33342staining method.8Previously added SP60012510μM,30minutes later, Neural stem cells are chronic incubatedwith Aβ_1-405μm at each point in time（0h、12h、24h、48h）, Expression of Caspase-3specific activity calculated by colorimetric method. To detect the effects of TEA act on Aβ_1-40-induced Neural stem cells.9Because of Aβ_1-40-induced Neural stem cells JNK phosphorylation activated peaked at24h, Therefore, at this time point added TEA5mM, then using Western blotmethod to detect a status which is JNK phosphorylation of Aβ_1-40-induced Neural stem cells.Results:1Single neuron in hippocampus of neonatal rats in freshly isolated stem cellsshowed bright round, at the third passage visible culture larger neurosphere formation fluid, thepositive expression of Nestin protein marker of Neural stem.2Effect of TEA on Aβ_1-40-inducedNeural stem cells’s survival rate, apoptosis rate and Caspase-3activity.2.1The MTT assayTEA survival of Aβ_1-40-induced Neural stem cells. The results show: Aβ_1-40group of Neural stemcells at different time points, cell significantly reduced （p＜0.05）, and over time prolong cellsurvival decreased even more obvious. Aβ_1-40+TEA group compared with Aβ_1-40group of cellssurvival was significantly increased （p＜0.05）, the control group and the TEA Neural stem cellsdid not change significantly （p＞0.05）.2.2Hoechst33342method was used to observe theoccurrence of TEA on the apoptosis of Neural stem cells induced by Aβ_1-40. The results show:Under a fiuorescence microscope Neural stem cells of Aβ_1-40group which some nuclei werehyperchromatic chunky48h after, for the typical apoptotic morphological changes. Apoptosisrate is20.3%±0.6%,（p＜0.01）. Aβ_1-40+TEA group than Aβ_1-40group of apoptosismorphological changes. Apoptosis rate is11.6%±0.4%, The difference was significant （p＜0.01）.2.3Colorimetric detection TEA neural stem cells of Aβ_1-40-induced Caspase-3activitychanges. The results show: Aβ_1-40+TEA group at each time point can reduce the expression ofCaspase-3（p＜0.05）, special in24h, this trend is more obvious （p＜0.01）. Blank control groupand TEA control group at all time points Caspase-3specific activity did not change significantly.3JNK and SP600125of JNK selective blocking agent on Aβ_1-40-induced Neural stem cellsphosphory-lation changes.3.1Western blot method to detect Aβ_1-40-induced Neural stem cellsof JNK expression of phosphorylated.The results show: Neural stem cells were incubated Aβ_1-405μM48h, Different time points the phosphorylation of JNK were expressed. JNK phosphorylation level gradually increase with time prolonging, and reached a peak at24h （p＜0.01）, andJNK blocker SP600125can significantly interrupt the activation.3.2MTT assay survival ofSP600125on Aβ_1-40-induced Neural stem cells. The results show: Aβ_1-40+SP600125group cansignificantly reduce Neural stem cells death, the survival of neural stem cells from72.7%±3.2%,50.35%±2.7%,42.7%±7.3%, respectively increased to92.6%±0.70%,76.1%±0.73%,67.0%±0.64%,（p＜0.01）.3.3Using Hoechst33342method to detect SP600125Neural stem cells apoptosis of Aβ_1-40-induced. The results show:Aβ_1-40+SP600125group cansignificantly reduce the incidence of Neural stem cells after Aβ_1-405μM incubated apoptisis. Theapoptotic rate of SP600125from20.3%±0.6%to9.6%±1.3%（p＜0.01）, SP600125group nosignificant changes. SP600125itself does not affect cell apoptosis.3.4Colorimetry to detectSP600125on Aβ_1-40-induced Caspase3activity changes of neural stem cells. The results show:Examining the role of JNK inhibitor SP600125, Aβ_1-40+SP600125group can significantlyreduce the activation of caspase-3, Aβ_1-40group12.46±0.47)pmol/h/μg decreased to （5.09±0.40） pmol/h/μg （n=3，p＜0.01）.4TEA effect of stem cells on the expression of JNKphosphorylation of Aβ_1-40-induced. The results show: Aβ_1-40+TEA group was significantlylower （p＜0.01） compared with Aβ_1-40group of JNK phosphorylation expression.Blank controlgroup and the TEA group Neural stem cells JNK phoshorylation showed no significant change（p＞0.05）.Conclusion：1Potassium channel blocker TEA can significantly mitigate Aβ_1-40-inducedNeural stem cells death,increased the survival of neural stem cells, significantly decreased theincidence of apoptdsis.2JNK signal transduction pathways involved in apoptosis signaltransduction, has an important role in the Aβ_1-40-induced Neural stem cells damage.3JNKspecific inhibitor SP600125inhibit Aβ_1-40-induced Neural stem cells death,has a significantprotective effect of Aβ_1-40-induced Neural stem cells toxicity.4There are certain connectionbetween potassium channel Aβ_1-40induces the activation and phosphorylation of JNK in both,and induced the apoptosis related proteins downstream.更多还原