【作者】 赵雪莲；

【导师】 毕建忠；

【作者基本信息】 山东大学 ， 神经病学， 2015， 硕士

【摘要】 背景：阿尔茨海默病(Alzheimer’s disease, AD)是一种以智能衰退、人格改变为主要临床表现的慢性中枢神经系统变性疾病,病程进行性发展。病理改变主要集中在颞、前额和顶叶,其中以海马改变最为明显,主要病理学特征为胞内过度磷酸化的tau蛋白所形成的神经元纤维缠结(neurofibrillary tangles. NFTs), β淀粉样蛋白(β-amyloid protein, Aβ)异常蓄积形成的老年斑(senile plaques, NPS)以及选择性神经元和突触脱失。在早发性家族性AD患者中涉及淀粉样前体蛋白(β-amyloid precursor protein, APP)、早老素1(presenilin 1, PS1)、早老素2(presenilin 2, PS2)基因突变。但是AD发病的分子机制在很大程度上还不清楚,在神经元纤维缠结形成前细胞已经出现能量代谢障碍、ROS生成增加,说明线粒体功能障碍以及氧化应激是AD的早期事件,目前几种假说主要围绕AD发生发展的早期事件进行研究,旨在为该疾病提供一种早期有效的临床干预治疗。自噬是细胞清除损伤的细胞器和蛋白质的分解代谢过程,通过这一过程有助于细胞适应本身代谢需要和实现细胞器的更新。自噬主要包括巨自噬、微自噬和分子伴侣介导的自噬三种形式,按自噬是否具有选择性又可分为选择性和非选择性自噬。线粒体是真核细胞内进行能量转化的场所,在供给细胞能量的同时可以产生活性氧(ROS),过多的ROS可以引起蛋白质,核酸和脂肪酸过氧化,造成细胞损伤,因此及时清除损伤的线粒体对维持细胞正常生理功能具有重要作用。当细胞在饥饿、氧化应激、衰老等情况下,可以使细胞内线粒体发生去极化,去极化的线粒体被选择性的包裹进自噬体并与溶酶体结合,从而使损伤的线粒体得以清除的过程称为线粒体自噬。线粒体自噬属于选择性自噬。有研究表明,AD患者大脑神经元内存在线粒体数量减少、嵴破坏,线粒体功能障碍。Ap可以抑制细胞呼吸链和三羧酸循环的关键酶,使线粒体功能障碍,线粒体膜电位下降,线粒体肿胀、颗粒性状发生改变、ATP生成减少,ROS增多,造成细胞损伤。线粒体靶向丝氨酸/苏氨酸激酶PTEN诱导激酶1(PTEN inducible kinase 1, PINK1)和E3泛素连接酶Parkin通过调节膜电位,嵴结构,钙稳态,线粒体DNA的完整性和呼吸活动等一些生理过程来维持线粒体完整性。在正常的线粒体中,PINK1被迅速降解,当线粒体膜电位去极化后,PINKl才以电压依赖的方式在线粒体外膜上稳定表达。PINK1/Parkin途径是自噬相关清除不正常线粒体的关键。根据以上背景所述,本研究主要探讨在AD细胞模型中是否有线粒体自噬异常,线粒体自噬的作用机制是什么,PINK1和Parkin是否参与了线粒体自噬的调节。本研究旨在对AD发病早期事件——线粒体自噬的研究,为AD的早期临床干预及防治提供新的思路及方法。目的：探讨阿尔茨海默病细胞模型20E2中是否存在线粒体自噬异常,线粒体自噬的作用机制以及PINK1和Parkin是否参与了线粒体自噬的调节。为AD的早期临床干预提供新思路及方法。方法：1.将20E2细胞(稳定转染APPsw的HEK293细胞)作为实验组,HEK293细胞作为对照组在相同的培养条件下培养。2.应用ELISA法检测细胞上清Aβ1-40水平、Western blotting检测APP蛋白表达水平,确定20E2细胞是否建模成功。3.电镜下观察细胞内线粒体。4.JC-1检测两种细胞线粒体膜电位。5.流式细胞仪检测两种细胞凋亡情况。6. Western blotting检测LC3-Ⅱ、PINK1、Parkin表达水平。7.免疫荧光观察Parkin表达及线粒体定位情况。8. PINK1 siRNA处理细胞,Western blotting检测自噬相关蛋白LC3-Ⅱ表达水平。1.与HEK293细胞相比,20E2细胞APP蛋白、Aβ1-40表达增加(P<0.05)。2.20E2较HEK293细胞线粒体肿胀、嵴消失、空泡化明显,线粒体膜电位下降。3.20E2细胞与HEK293细胞凋亡率无明显差别(P>0.05)。4.与HEK293细胞相比,20E2细胞LC3-Ⅱ、 PINK1、Parkin表达增加(P<0.05)。5.免疫荧光示20E2较HEK293细胞Parkin表达增加,线粒体定位明显。6.经PINK1 siRNA干扰后20E2细胞LC3 Ⅱ表达减少,说明PINK1/Parkin途径参与了20E2细胞线粒体自噬调节。结论：阿尔茨海默病细胞模型20E2线粒体形态改变明显、膜电位下降,线粒体自噬增加,PINK1 Parkin表达增加,经PINK1 siRNA处理后20E2细胞自噬较前减弱,研究结果表明AD细胞模型20E2这些改变可能通过PINK1、Parkin途径引起线粒体自噬增加。更多还原

【Abstract】 Background:Alzheimer’s disease (AD), is a chronic degenerative disease of the central nervous system, cognitive function decline and personality change as the main clinical manifestation, Pathological changes are mainly concentrated in the temporal, prefrontal and parietal lobe, and the most obvious change is in the hippocampus. Neurofibrillary tangles are formed with hyperphosphorylated tau protein, and senile plaques(NPS) are formed with β-amyloid protein(Aβ) abnormal deposit. NPS, Ap and the selectivity of neurons and synapses loss are the major pathological features of AD. amyloid-β protein precursor (APP) and presenilin (PS1) 1 and presenilin 2 (PS2) genes mutations are involved in early-onset familial of AD. However, the molecular mechanism of the pathogenesis of AD is unclear, prior to the formation of neurofibrillary tangles cell energy metabolism and ROS generation increased has occurred, indicating oxidative stress and mitochondrial dysfunction is an early event in AD. Currently several hypotheses are focus on the early events in the development of AD research, the purpose is to provide effective clinical intervention for an early disease.Autophagy is a catabolic process to eliminate damaged organelles and protein, and through this process helps the cell adapt to their metabolism needs and achieve cell organelles updates. Autophagy includes macroautophagy, microautophagy and chaperon-mediated autophagy three forms and according to the selective autophagy also divided into selective and non-selective autophagy. Mitochondria are the energy conversion sites in eukaryotic cells, can produce reactive oxygen species (ROS) at the same time, excessive ROS can cause the protein, nucleic acid and fatty acid peroxidation, cause cell damage, therefore removal of damaged mitochondria in time plays an important role in maintaining the normal physiological functions of cells. When the cells under starvation, ROS, aging and other circumstances, can make the mitochondria to the depolarization, depolarization of mitochondria are selectively packaged into autophagosomes and combined with lysosomes, so that the process to clear the damage mitochondria known as mitophagy. Mitophagy are selective autophagy. Studies have shown that, in AD patients brain mitochondrial dysfunction, cristae damaged, and the number of mitochondrial decreased. Aβ can inhibit cell respiratory chain and the key enzymes of the tricarboxylic acid cycle, and lead to mitochondrial dysfunction, decrease the mitochondrial membrane potential, mitochondrial swelling, change the granular characters, decrease the production of ATP, ROS increase, causing cell injury.Mitochondrial targeting serine/threonine kinase PTEN inducible kinase 1 (PINK1) and E3 ubiquitin ligase Parkin by regulating the membrane potential, cristae structure, calcium homeostasis, mitochondrial DNA integrity, respiratory activities and a number of physiological process to maintain mitochondrial integrity. In normal mitochondria, PINK1 is degraded rapidly, when the mitochondrial membrane potential depolarizing, PINK1 with a voltage dependent manner stable expression in the mitochondrial outer membrane. The PINK1/Parkin pathway is the key of autophagy related to remove abnormal mitochondrial.Based on the above background, this research mainly to investigate whether cell model of AD have abnormal mitophagy, what is the mechanism of mitophagy, and whether PINK1 and Parkin is involved in the regulation of mitophagy. The purpose of this study is to research on the early AD events -- mitophagy, provides the new ideas and methods for early clinical intervention and prevention of AD. Objective:This study is to investigate whether cell model of AD have abnormal mitophagy, what is the mechanism of mitophagy, and whether PINK1 and Parkin is involved in the regulation of mitophagy. The purpose of this study is to provides the new ideas and methods for early clinical intervention of AD.Methods:1. We used the 20E2 cells (HEK293 cells stably expressing Swedish mutant APP) as the experimental group, HEK293 cells were used as the control group, and they cultured under the same culture conditions.2. To determine whether the 20E2 cells model was successfully established, we detected the extracellular concentration of Aβ1-40 by ELISA kit, and the expression of APP protein level was detected by Western blotting.3. Observation of mitochondria in cells under electron microscopy.4. The mitochondrial membrane potential of both cells were detected by fluorescence probe JC-1.5. Detection of both cells apoptosis by flow cytometry.6. The expression levels of LC3-II, PINK1 and Parkin were detected by Western blotting.7. The expression of Parkin and it’s mitochondrial localization observed by Immunofluorescence.8. Cells treated with PINK1 siRNA, western blotting detected the expression levels of autophagy related protein LC3-II, PINK1. Results:1. Compared with HEK293 cells, the expression leves of APP protein and Aβ1-40 in 20E2 cells increased(P<0.05).2. Compared with HEK293 cells, mitochondrial swollen, cristae disappeared and vacuolization were obviously observed in 20E2, mitochondrial membrane potential decrease.3. Compared with HEK293 cells, the apoptosis rate showed no significant difference in 20E2 cell (P>0.05).4. Compared with HEK293 cells, the expression levels of LC3-Ⅱ, PINK1 and Parkin increased in 20E2.5. Compared with HEK293 cells, Immunofluorescence showed the expression of Parkin increased and mitochondrial localization was obvious in 20E26. The expression of LC3-Ⅱ reduced after PINK1 siRNA interference in 20E2 cell, indicating that PINK 1/Parkin pathway is involved in the regulation of mitophagy. Conclusion:In Alzheimer’s disease cell model 20E2, the mitochondrial morphology changed obviously and membrane potential of mitochondria declined. Mitophagy increased, the expression levels of PINK1, Parkin increased, autophagy reduced after siRNA treatment, and these changes may cause the increase of mitochondrial autophagy through PINK1 and Parkin pathway.更多还原