丁内酯衍生物对阿尔茨海默氏病细胞模型的作用及其相关分子机制的研究

发布时间：2018-07-12 10:08

本文选题：阿尔茨海默氏病 + Aβ　；参考：《山东大学》2012年博士论文

【摘要】：研究背景和研究目的阿尔茨海默氏病(Alzheimer disease, AD)是神经系统进行性变性疾病,是痴呆最常见的病因。临床上表现为记忆障碍、失语、失用、失认、视空间能力损害、抽象思维和计算力损害、人格和行为的改变等。全球AD患者逐年增多,在发达国家老年人群中,已成为导致死亡的第四位原因。AD的发病机制迄今不明,亦无特效治疗方法。因此明确AD发病机制,针对其不同环节,寻求防治AD的新方法仍是目前亟需解决的重大问题。 AD可见颞、顶及前额叶萎缩。其主要病理特征：老年斑(senile plaques, SPs)、神经原纤维缠结(neurofibrillary tangles, NFTs)、颞叶和海马皮质等部位神经元丢失、颗粒空泡变性、血管淀粉样变。其中老年斑是含有β-淀粉样蛋白(β-amyloid protein, Aβ)、早老素1、早老素2、a1抗糜蛋白酶、载脂蛋白E、α2巨球蛋白和泛素等的细胞外沉积物,神经原纤维缠结是含有磷酸化tau蛋白(一种微管相关蛋白)和泛素的细胞内沉积物,SPs、NFTs和神经元丢失为AD特征性病理改变。Ap异常聚集是各种原因诱发AD的共同通路,由此研究Ap对神经细胞的毒性作用和损伤机制以及阻止和消除Ap沉积已成为研究AD发病机制的重要靶点。自噬(autophagy)是真核细胞中广泛存在的降解/再循环系统。自噬可分为三种主要方式：大自噬、小自噬和分子伴侣介导的自噬,大自噬是最常见的自噬形式。自噬体-溶酶体系统是近年来研究Ap沉积的新方向。在发病早期的AD患者和8周龄的APP/PS1转基因AD鼠脑内,均发现有大量的自噬体存在。细胞内的Aβ可以由细胞内自噬体-溶酶体中的β淀粉样前体蛋白(β-amyloid precursor protein, APP)裂解产生,细胞外的Aβ亦可以激活自噬过程清除异常蛋白,但自噬的过度激活亦可导致细胞死亡。因此自噬在AD发病机制中的具体作用仍有待进一步研究。之前的研究发现一种新型丁内酯衍生物3BD0(3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one)可以抑制血管内皮自噬功能,防止细胞空泡化,抑制血管内皮细胞凋亡,防止细胞老化。那么在神经细胞中尤其是AD细胞模型中,是否也有同样的作用?是否可以通过调节自噬对抗Aβ的毒性、减少Aβ的沉积来治疗AD呢? 依据上述背景,本研究以化学小分子为工具,干预了AD细胞模型的自噬过程,进而研究自噬在AD发病机制中的作用并寻找参与调控自噬的关键因子,这有助于明确AD的发病机制、寻找新的AD治疗靶点和开发新型靶向药物。研究内容 1.研究外源性的Aβ25-35对PC12细胞的毒性作用及是否能够诱导细胞自噬。 2.研究3BDO是否可以保护PC12细胞拮抗Aβ的细胞毒性作用及作用机制。 3.研究3BDO能否抑制AD转基因细胞模型20E2细胞Aβ的生成以及其作用机制。 4.研究3BDO抑制自噬的分子机制。研究方法 1.AD细胞模型的构建、培养：PC12细胞(大鼠嗜铬神经瘤细胞)和SH-SY5Y细胞(人神经母细胞瘤细胞)正常培养,加入老化处理的Aβ25-35构建AD细胞模型一；20E2细胞(HEK293细胞稳定转染瑞典突变APP基因)为AD细胞模型二。 2.细胞存活率的检测：利用SRB方法检测细胞存活率。 3.细胞凋亡的检测：Hoechest33258染色结合荧光显微镜,观察细胞核凝集。 4.细胞自噬的检测： 4.1通过吖啶橙染色在荧光显微镜下观察细胞中酸性膜泡的数量及分布。 4.2通过检测LC3-Ⅱ蛋白水平检测自噬是否被诱导。 5.ROS检测：利用荧光探针(DCHF)结合激光扫描共聚焦显微技术进行。 6. Na+K+-ATP酶活性检测：按照试剂盒说明检测Na+K+-ATP酶活性。 7.线粒体膜电位检测：利用荧光探针(JC-1)结合激光共聚焦显微技术进行。 8.细胞外Aβ1-40、Aβ1-42浓度检测：利用ELISA方法参照说明书进行检测。 9. FLJ11812的表达分析：利用反转录PCR(RT-PCR)和琼脂糖凝胶电泳相结合,检测FLJ11812的水平变化。 10.细胞蛋白表达水平检测：使用western blot法检测LC3-II, p70s6k, p-p70s6k,p62, APP, IDE, NEP的蛋白水平。研究结果 1丁内酯衍生物3BDO保护PC12细胞拮抗Ap的细胞毒性作用及其作用机制 1.1Aβ25-35外源性干预PC12细胞可以引起细胞存活率降低,并呈剂量和时间依赖性。 1.23BDO预处理1小时后可以减少Aβ25-35引起的细胞存活率的降低,发挥保护作用。60μM和120μM的3BDO预处理1小时后可以抑制10μM Aβ引起的细胞存活率的下降。 1.33BDO可以减少Aβ25-35引起的ROS水平的增加。10μM Aβ处理2或4小时后,细胞内ROS水平较对照组明显增加,3BDO预处理1小时后可以减少Ap引起的细胞内ROS的累积。 1.43BDO可以修复Aβ25-35引起的Na+K+-ATP酶活性的损伤。10μM Aβ25-35处理细胞4h后,Na+K+-ATP酶活性受到了抑制。120μM3BDO单独处理细胞时,该酶活性与对照组相比没有明显改变。120μM的3BDO预处理细胞1h再加入Aβ后,与Ap组相比Na+K+-ATP酶活性明显上升。 1.53BDO对Aβ25-35引起的细胞线粒体膜电位的改变没有明显影响。10μM Aβ25-35处理细胞4h后,细胞线粒体膜电位与对照组相比明显升高。而3BDO和Aβ共处理组线粒体膜电位与Ap处理组相比没有明显恢复。这表明3BDO对Aβ25-35引起的细胞线粒体膜电位的改变没有明显影响。 1.63BDO拮抗Ap细胞毒性的机制研究： 1.6.110μM Aβ25-35、120μM3BDO对细胞凋亡没有明显影响。但是3BDO可以减少Aβ25-35引起的细胞内酸性膜泡的增加。我们利用Hoechst染色法检测细胞有无凋亡现象。10μM Aβ25-35、120μM3BDO及共处理组细胞凋亡与对照组相比没有明显差别,说明低浓度的Aβ25-35引起的细胞存活率的下降并不是由于增加细胞凋亡引起的。随后我们利用吖啶橙染色观察细胞内酸性膜泡的变化来初步鉴定细胞自噬的变化。10μM Aβ25-35处理细胞4h后,细胞内酸性膜泡的累积与对照组相比明显增加,而3BDO预处理1h后,Aβ引起的细胞内酸性膜泡的累积被抑制了 1.6.23BDO可以减少Aβ25-35引起的LC3-Ⅱ的增加。LC3-Ⅱ已经作为一种自噬小体特异性标记物被广泛应用于自噬的检测。为了研究Aβ25-35和3BDO对于自噬小体数量的影响,我们用Western blot法检测了细胞中LC3加工的情况。结果显示3BDO能够显著地抑制Aβ引起的LC3-Ⅱ的积累。 23BDO抑制AD转基因细胞模型20E2细胞外Aβ的生成及其作用机制的研究 2.13BDO可以减少SH-SY5Y细胞及20E2细胞外Aβ1-40的浓度。我们提取含有同一数量级细胞的细胞培养液上清进行ELISA检测。实验结果发现,3BDO可以抑制SH-SY5Y细胞、20E2细胞Aβ1-40的产生,而对HEK293细胞没有明显影响。 2.23BDO可以降低20E2细胞外Aβ1-42的水平,而对SH-SY5Y细胞及HEK293细胞外Aβ1-42无明显影响。 2.33BDO对细胞内APP水平无明显影响。我们利用Western blot方法检测了细胞内APP水平有无变化。3BDO处理前后20E2和SH-SY5Y细胞中APP水平没有变化,说明3BDO并不是通过降低APP的水平来减少细胞外Ap的水平。 2.43BDO可以抑制HEK293细胞、SH-SY5Y细胞、20E2细胞LC3-Ⅱ蛋白水平。120μM3BD0处理24小时后,三种细胞的LC3-Ⅱ蛋白水平均受到了抑制,20E2细胞、SH-SY5Y细胞p62水平升高,进一步说明了3BDO抑制了这两个细胞内的自噬水平。我们初步推断3BDO可能通过抑制自噬抑制了其内Aβ的生成。 2.53BDO可以升高细胞内胰岛素降解酶(IDE)及肾胰岛素残基溶解酶(NEP)水平。120μM3BD0处理24小时后,20E2细胞中NEP和IDE含量与对照组相比水平增加,我们推断3BDO还可能通过增加Aβ降解酶增加Aβ的清除的方式减少Ap的胞外累积。 33BDO抑制自噬的分子机制研究 3.13BDO是以mTOR依赖的方式调控Aβ诱导的自噬过程。mTOR介导的信号通路是自噬过程的经典通路,mTOR是其中的关键分子。我们利用Western blot法检测mTOR下游底物p70S6K的磷酸化水平的变化检测其活性。同时利用雷帕霉素作为阳性对照处理细胞。结果显示雷帕霉素及Aβ均可抑制mTOR活性激活自噬,60μM及120μM3BDO可通过激活mTOR活性抑制自噬。 3.23BDO可以升高Aβ引起的SH-SY5Y细胞内FLJ11812基因水平的下降。10μMAβ25-35处理SH-SY5Y细胞4小时后,FLJ11812cDNA水平下调,3BDO预处理1小时后可以上调其表达。说明FLJ11812可能参与了3BDO对抗Aβ毒性的作用机制中。 3.3过表达FLJ11812后,细胞整体自噬水平受到了抑制。在正常实验组及过表达FLJ11812的实验组中,3BDO都可以抑制Aβ引起的LC3-Ⅱ的增加。在过表达FLJ11812之后细胞内的LC3-Ⅱ水平与对照组相比均减少。在正常实验组及过表达FLJ11812的实验组中,3BDO都可以上调Aβ引起的p62的降低,进一步说明了3BDO可以抑制自噬。实验结果说明过表达FLJ11812可以抑制细胞内LC3-Ⅱ的积累,上调p62水平,抑制自噬过程。 3.4利用siRNA干扰掉FLJ11812的表达后,自噬标记蛋白LC3-Ⅱ含量无明显变化。结论 1. Aβ可以产生细胞毒性作用,减少细胞存活率,呈时间和剂量依赖性。 2.3BDO可以抑制Ap引起的细胞存活率的降低,减少Ap引起的细胞内ROS水平的增加,修复Aβ引起的细胞内Na+K+-ATP酶活性的损伤,但对Ap引起的细胞线粒体膜电位的改变没有明显影响。 3.3BDO可以增加细胞存活率是通过抑制细胞自噬而对细胞凋亡没有明显影响。 4.3BDO可以可能通过抑制AD转基因细胞20E2细胞自噬水平减少Ap的生成,并且可以抑制20E2细胞内Ap降解酶NEP的表达增加Aβ的降解,减少细胞外Aβ1-40、Aβ1-42的产生。 5.3BDO是通过上调mTOR信号通路抑制自噬,同时FLJ11812在3BDO调控自噬的过程中发挥了重要作用,3BDO通过上调FLJ11812的表达抑制自噬。
[Abstract]:Research background and purpose
Alzheimer disease (AD) is a progressive neurodegenerative disease of the nervous system. It is the most common cause of dementia. It is characterized by memory disorders, aphasia, loss of use, apathy, visual impairment, abstract thinking and computational power impairment, and changes in personality and behavior. The number of AD patients worldwide increased year by year in the elderly population of developed countries. The pathogenesis of.AD, the fourth cause of death, has so far been unknown and there is no special therapeutic method. Therefore, it is still a major problem to be solved to find a new mechanism for the pathogenesis of AD and to seek a new method to prevent and control AD.
AD shows temporal, top and prefrontal atrophy. The main pathological features are senile plaque (senile plaques, SPs), neurofibrillary tangles (neurofibrillary tangles, NFTs), loss of neurons in the temporal and hippocampal cortex, granular vacuolation, and amyloidosis of blood vessels. The senile plaques contain beta amyloid (beta -amyloid protein, A beta), early Elochin 1, proin 2, A1 anti chymotrypsin, apolipoprotein E, alpha 2 giant globulin and ubiquitin and other extracellular sediments, neurofibrillary tangles containing phosphorylated tau protein (a microtubule related protein) and ubiquitin intracellular sediments, SPs, NFTs, and neuronal loss of AD characteristic pathological changes of.Ap abnormal aggregation are various causes inducing AD Therefore, studying the toxicity and damage mechanism of Ap to neurons and preventing and eliminating Ap deposition have become an important target for studying the pathogenesis of AD.
Autophagy (autophagy) is a widespread degradation / recirculation system in eukaryotic cells. Autophagy can be divided into three main ways: autophagy, small autophagy and autophagy mediated by chaperone. Autophagy is the most common form of autophagy. Autophagosome system is a new direction for the study of Ap deposition in recent years. In early onset AD patients and 8 weeks of age A large number of autophagosomes are found in the brain of APP/PS1 transgenic AD mice. The intracellular A beta can be produced by the lysis of beta amyloid precursor protein (beta -amyloid precursor protein, APP) in the autophagosome, and the extracellular A beta can activate autophagy to clear the ISO protein, but the excessive activation of autophagy can also lead to cells. Therefore, the specific role of autophagy in the pathogenesis of AD remains to be further studied.
Previous studies have found that a new butylene derivative, 3BD0 (((2-nitrophenoxy) methyl) -dihydrofuran-2 (3H) -one, can inhibit autophagy, prevent vacuolization of cells, inhibit apoptosis of vascular endothelial cells and prevent cell aging. Then, it is also the same in the neural cells, especially in the AD cell model. Can we treat AD by regulating autophagy against the toxicity of A beta and reducing the deposition of A beta?
Based on the above background, this study used small molecules as a tool to intervene the autophagy process of the AD cell model, and then study the role of autophagy in the pathogenesis of AD and find the key factors involved in the regulation of autophagy. This is helpful to clarify the pathogenesis of AD, to find new targets for the treatment of AD and to develop new target drugs.
research contents
1. to study the toxic effects of exogenous A beta 25-35 on PC12 cells and whether they can induce autophagy.
2. to study whether 3BDO can protect PC12 cells against the cytotoxic effect of A beta and its mechanism.
3. to study whether 3BDO can inhibit the production of A beta in 20E2 cells of AD transgenic cells and its mechanism.
4. study the molecular mechanism of 3BDO inhibition of autophagy.
research method
The construction of 1.AD cell model, culture: normal culture of PC12 cells (rat chromaffin neuroma cells) and SH-SY5Y cells (human neuroblastoma cells), adding the aging treated A beta 25-35 to construct a AD cell model, and 20E2 cells (HEK293 cells stably transfected with Swedish mutant APP base) is a AD cell model of two.
2. detection of cell viability: SRB assay was used to detect cell viability.
3. apoptosis detection: Hoechest33258 staining combined with fluorescence microscopy to observe nuclear agglutination.
4. cell autophagy: detection of autophagy:
4.1 the number and distribution of acid membrane vesicles in the cells were observed under acridine orange staining under fluorescence microscope.
4.2 to detect whether autophagy is induced by detecting the level of LC3- II protein.
5.ROS detection: using fluorescence probe (DCHF) combined with laser scanning confocal microscopy.
6. Na+K+-ATP enzyme activity assay: Na+K+-ATP enzyme activity was detected according to the kit.
7. mitochondrial membrane potential detection: using fluorescence probe (JC-1) combined with laser confocal microscopy.
8. detection of extracellular A beta 1-40 and A beta 1-42 concentration: ELISA method and reference manual for detection.
9. expression analysis of FLJ11812: reverse transcription PCR (RT-PCR) and agarose gel electrophoresis were used to detect the level of FLJ11812.
10. detection of cellular protein expression: the protein levels of LC3-II, P70S6K, p-p70s6k, p62, APP, IDE and NEP were detected by Western blot.
Research results
1 butylene derivative 3BDO protects PC12 cells against Ap cytotoxicity and its mechanism
Exogenous 1.1A beta 25-35 interfered with PC12 cells, which could induce cell viability to decrease in a dose and time dependent manner.
1.23BDO pretreatment could reduce the decrease of cell survival rate caused by A beta 25-35 after 1 hours of pretreatment, and the survival rate of cells induced by 10 u M A beta could be inhibited after 1 hours of 3BDO preconditioning with protective effect of.60 Mu and 120 mu M.
1.33BDO can reduce the level of ROS induced by A beta 25-35. After 2 or 4 hours of.10 - M A beta treatment, the intracellular ROS level is significantly higher than that of the control group. 3BDO preconditioning can reduce the accumulation of ROS in the cells caused by Ap after 1 hours.
When 1.43BDO could repair the Na+K+-ATP enzyme activity induced by A beta 25-35, the activity of.10 mu M A beta 25-35 treated cell 4h, the activity of Na+K+-ATP enzyme was inhibited by.120 micron M3BDO alone, and the activity of the enzyme did not significantly change the activity of.120 micron M pretreated cells, and the activity of the enzyme was obviously higher than that of the control group. Rise.
The mitochondrial membrane potential of cell mitochondrial membrane potential induced by A beta 25-35 was not significantly affected by.10 mu M A beta 25-35 treated cell 4h, and the mitochondrial membrane potential of cell mitochondria was significantly higher than that of the control group. The mitochondrial membrane potential of 3BDO and A beta co processing group was not significantly restored to the Ap treatment group. This indicates that 3BDO has a mitochondrial membrane caused by A beta 25-35. The change of potential was not significantly affected.
The mechanism of 1.63BDO to antagonize the toxicity of Ap cells:
1.6.110 micron M A beta 25-35120 M3BDO has no obvious effect on cell apoptosis. But 3BDO can reduce the increase of intracellular acidic vesicles induced by A beta 25-35. We use Hoechst staining method to detect cell apoptosis in.10 u M A beta 25-35120 micron M3BDO and there is no significant difference in cell apoptosis compared with the control group, indicating low concentration. The decrease of cell survival rate caused by A beta 25-35 was not caused by increasing cell apoptosis. Then we used acridine orange staining to observe the changes in intracellular acid vesicles to preliminarily identify the changes in cell autophagy by.10 mu M A beta 25-35 treatment of cell 4h, the accumulation of intracellular acid vesicles was significantly increased compared with the control group, while 3BDO was predisposed. After 1h, the accumulation of intracellular acidic membrane vesicles induced by A beta was inhibited.
1.6.23BDO can reduce the increase of LC3- II induced by A beta 25-35.LC3- II has been widely used as an autophagic specific marker for autophagy detection. In order to study the effect of A beta 25-35 and 3BDO on the number of autophagic corpuscles, we detected LC3 addition in the cells by Western blot method. The results showed that 3BDO could be significant. Inhibition of the accumulation of LC3- II induced by A beta.
Inhibitory effect of 23BDO on the formation of extracellular 20E2 A in AD transgenic cell lines and its mechanism
2.13BDO can reduce the concentration of A beta 1-40 outside the SH-SY5Y cells and 20E2 cells. We extract the cell culture supernatant containing the same number of cells for ELISA detection. The results showed that 3BDO could inhibit the production of A beta 1-40 in SH-SY5Y cells and 20E2 cells, but had no obvious effect on HEK293 cells.
2.23BDO can reduce the level of extracellular A beta 1-42 in 20E2 cells, but has no significant effect on A cells and SH-SY5Y cells in SH-SY5Y cells and HEK293 cells.
2.33BDO had no obvious effect on the intracellular APP level. We used Western blot to detect the level of APP in cells without change of.3BDO before and after.3BDO treatment. The APP level in 20E2 and SH-SY5Y cells did not change, indicating that 3BDO did not reduce the level of extracellular Ap by reducing APP.
2.43BDO can inhibit HEK293 cells, SH-SY5Y cells, and 20E2 cell LC3- II protein level.120 mu M3BD0 after 24 hours treatment, the level of LC3- II protein of three cells is inhibited, 20E2 cells, SH-SY5Y cells p62 level, further demonstrate that 3BDO inhibits the level of autophagy within the two cells. We preliminarily infer that it may pass inhibition. Autophagy inhibits the formation of A beta in it.
2.53BDO could increase the level of intracellular insulin degrading enzyme (IDE) and renal insulin residue dissolving enzyme (NEP) level for 24 hours. After 24 hours, the content of NEP and IDE in 20E2 cells increased compared with that of the control group. We infer that 3BDO may also reduce the extracellular accumulation of Ap by increasing A beta degrading enzyme and increasing A beta clearance.
Study on the molecular mechanism of inhibition of autophagy by 33BDO
3.13BDO is a mTOR dependent manner to regulate the process of autophagy induced by A beta,.MTOR mediated signaling pathway is the classic pathway of autophagy. MTOR is the key molecule. We use Western blot to detect the activity of the phosphorylation level of p70S6K in the lower reaches of mTOR, and use rapamycin as a positive control treatment. Cells showed that rapamycin and A beta could inhibit mTOR activity and activate autophagy. 60 M and 120 M3BDO could inhibit autophagy by activating mTOR activity.
3.23BDO can increase the level of FLJ11812 gene in SH-SY5Y cells induced by A beta, and.10 u MA beta 25-35 can reduce the level of FLJ11812cDNA after 4 hours of SH-SY5Y cells. 3BDO preconditioning can increase its expression after 1 hours. It indicates that FLJ11812 may be involved in 3BDO antagonism to A beta toxicity.
3.3 after overexpression of FLJ11812, the level of autophagy was inhibited. In the normal experimental group and in the experimental group that overexpressed FLJ11812, 3BDO could inhibit the increase of LC3- II induced by A beta. The level of LC3- II in the cells after overexpression of FLJ11812 was reduced compared with the control group. In the normal experimental group and in the experimental group that overexpressed FLJ11812, 3BDO can all increase the decrease of p62 induced by A beta, and further demonstrate that 3BDO can inhibit autophagy. The results show that overexpression of FLJ11812 can inhibit the accumulation of LC3- II in cells, up-regulate the p62 level and inhibit the autophagy process.
3.4 there was no significant change in the content of autophagy labelled protein LC3- II after interfering with the expression of FLJ11812 by siRNA.
conclusion
1. A beta can produce cytotoxicity and reduce cell viability in a time and dose-dependent manner.
2.3BDO can inhibit the decrease of cell survival rate caused by Ap, reduce the increase of intracellular ROS level caused by Ap and repair the damage of intracellular Na+K+-ATP activity induced by A beta, but it has no significant effect on the changes of mitochondrial membrane potential induced by Ap.
3.3BDO can increase cell survival rate by inhibiting autophagy and has no obvious effect on cell apoptosis.
4.3BDO can reduce the formation of Ap by inhibiting the autophagy level of 20E2 cells of AD transgenic cells, and can inhibit the expression of Ap degrading enzyme NEP in 20E2 cells to increase the degradation of A beta, and reduce the production of A beta 1-40, A beta 1-42.
5.3BDO inhibits autophagy by up-regulating the mTOR signaling pathway, while FLJ11812 plays an important role in 3BDO regulation of autophagy, and 3BDO inhibits autophagy by up regulation of FLJ11812 expression.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

【参考文献】