腹膜透析清除脑内Aβ和改善阿尔茨海默病相关病理的作用和机制研究

发布时间：2018-04-25 22:38

本文选题：β-淀粉样肽 + 腹膜透析　；参考：《第三军医大学》2017年博士论文

【摘要】：背景和目的阿尔茨海默病(Alzheimer's disease, AD)是目前老年人中最主要的痴呆类型,是一种以进行性的记忆能力下降和认知功能障碍为特征的神经退行性疾病。AD病因复杂,迄今为止,其发病机制尚未阐述清楚。传统观点认为,AD是大脑自身的疾病。脑内β-淀粉样肽(Amyloid-beta,Aβ)沉积形成的Aβ斑块(Aβ plaque)是AD最主要的病理特征。目前占主流地位的Aβ级联假说认为,AD的主要发生机制是脑内Aβ的过度产生或清除障碍导致AP在脑内过度沉积,诱发神经炎症、Tau蛋白过度磷酸化、脑内氧化应激以及神经变性死亡,最终导致痴呆。但是,近年来认识到,AD相关病理不仅局限于大脑。研究发现,Aβ沉积同时存在于肠道、皮肤和心脏,且有可能影响心脏功能。我们和他人的系列研究也表明,如系统慢性感染、心功能不全、肝肾功能不全、慢性肺部感染等系统疾病与Aβ代谢或AD密切相关。提示系统脏器组织功能状态可能参与了 AD的发生发展,值得从系统角度来探讨AD的发生机制与防治策略。目前尚无有效预防或延缓AD进展的药物和方法。Aβ是当前AD防治研究的最为重要的靶点,近年来开展了一系列针对Aβ产生和清除的临床试验,如Aβ主动与被动免疫治疗,β或γ分泌酶抑制剂,但这些试验都未能取得预期疗效。临床试验失败的重要原因之一是引起严重副作用。在免疫治疗临床试验中,抗体或药物进入脑内,在与脑内Aβ直接作用同时,导致脑水肿、微出血、无菌性脑膜脑炎以及神经元兴奋性毒性等副作用。同时,大脑功能的正常运行需要一个高度稳定的内环境,外源性药物通过血脑屏障进入脑内,可改变大脑内环境稳态,从而存在诱发不良反应的潜在风险。避免药物进入脑内,通过清除血液Aβ来促进脑内Aβ外流和清除,可能是更为安全的有效Aβ清除策略。我们过去研究发现,机体外周脏器组织在清除脑内Aβ方面具有重要作用,提示增强Aβ外周清除能力有望成为一种安全有效的AD治疗策略,值得探讨。腹膜透析(peritoneal dialysis, PD)是常被用于清除血液以及脑内的有毒物质,有望成为新的Aβ外周清除措施。为此,本课题拟通过临床研究和动物实验来探讨腹腔透析清除血液Aβ清除,防治AD的有效性和可行性。这些研究的完成,有助于从系统性角度来理解AD发生机制,寻找有效防治措施。材料和方法1.收集初次接受腹膜透析的30例慢性肾病(Chronic Kidney Disease, CKD)患者透析前后的血液样本以及腹膜透析液样本,检测并比较三种样本内Aβ水平。2.构建小鼠腹膜透析模型。本课题统一使用来自第三军医大学动物实验室的雌性C57野生型小鼠(Wt小鼠)为空白对照组,来自美国Jackson实验室的APPswe/PS 1 dE9(APP/PS 1)转基因小鼠为腹膜透析治疗组。实验组中预防组设置为从6月龄开始透析到7月龄终止,治疗组设置为9月龄开始透析到10月龄终止;接受置管手术但不透析的APP/PS1小鼠为阴性对照组。3.构建小鼠脑内微透析模型。APP/PS1小鼠和Wt小鼠来源同上。选用9月龄APP/PS1小鼠进行微透析实验,先按上述步骤建立腹膜透析模型。微透析实验选择海马区进行置管,植入微透析探针。手术10天后,待小鼠伤口愈合,拔出针芯植入半透膜在腹膜透析过程中进行微透析实验。阴性对照为Wt小鼠。4.取材。过量6%水合氯醛麻醉小鼠后打开小鼠胸腔,心尖取血,0.85%的生理盐水经心尖穿刺灌注,剪开右心耳。沿中线切开,取脑称重。右侧大脑半球于4%多聚甲醛固定,用作免疫染色;左侧大脑半球切取厚度约为100um左右冠状片子用于Golgi染色,其余部分液氮速冻后,保存于-80℃,用作生化分析。5.AD样病理改变染色。6E10、4G8和刚果红染色检测Aβ斑块。6E10、4G8分别与Iba-1共染检测小胶质细胞吞噬AP的能力。pS396染色检测Tau磷酸化水平。CD45与GFAP用于检测胶质细胞增生。6. ELISA检测。取适量脑组织,TBS匀浆。10级强度超声,100,000×g4℃离心1小时,取上清液,为可溶性提取物;管底组织继续加入200μl 2%SDS液,10级强度超声,100,000×g 4℃离心1小时,取上清液,为SDS提取物。管底组织继续加入200μl 70%FA液,10级强度超声处理30秒后,100,000×g4℃离心1小时,取上清液,为FA提取物。采用ELISA试剂盒检测小鼠血液,微透析后液体以及TBS、SDS和FA提取物中的Aβ40和Aβ42水平。脑匀浆中炎症因子如TNF-α、IFN-γ、IL-1β、IL-6、IL-4和IL-10的水平也用ELISA法检测。7.免疫印迹。取部分脑组织加入适量RIPA-B裂解液溶解,超声处理后,于10,00O×g 4℃离心30分钟。使用BCA法测定样品总蛋白浓度。配制合适浓度的电泳胶进行免疫印迹实验,本实验主要使用抗 Aβ、APP-C、sAPP、pS199、pS396、PSD93、PSD95、synapsin-1、synaptophysin、LRP-1和RAGE抗体检测目标蛋白,使用抗Actin抗体作为内参。8. Golgi染色。本实验选用FD Rapid GolgiStain Kit进行Golgi染色,实验方法严格按照试剂盒的说明进行。9.行为学与电生理实验。预防组与治疗组APP/PS1小鼠都进行Y-迷宫与旷场实验,治疗组动物进一步通过电生理实验检测腹膜透析对APP/PS1小鼠海马区长时程增强(Long-term Potentiation,LTP)的影响。结果1.腹膜透析降低CKD病人以及APP/PS1小鼠血液中Aβ水平。2.微透析实验表明,腹膜透析在降低小鼠血液中Aβ的同时可以迅速降低小鼠脑内ISF中的Aβ水平,并且小鼠血液与ISF中的Aβ变化趋势相关。3.在预防实验和治疗实验中,腹膜透析均能降低APP/PS1小鼠脑内Aβ水平。4.腹膜透析小鼠脑内TNF-α、IFN-γ和IL-6水平均明显降低IL-10水平显著升高,且其脑内异常活化的小胶质细胞和星形胶质细胞显著减少。5.腹腔透析提高脑内小胶质细胞对Aβ的吞噬能力有。6.腹膜透析改善APP/PS1小鼠脑内AD相关病理:海马神经元死亡减少,tau蛋白磷酸化水平(pS199和pS396)降低,轴突密度增加,神经元突触棘数量增加。7.腹膜透析改善APP/PS1小鼠认知功能:治疗组小鼠行为学结果,包括Y-迷宫(自发交替反应与新臂识别实验)和旷场实验检测结果得到显著改善。电生理实验提示腹膜透析显著改善APP/PS1小鼠海马区LTP。讨论本研究中,我们发现腹膜透析可有效降低血液与ISF中的可溶性Aβ水平。在为期一个月的透析后,APP/PS1小鼠脑内的Aβ负荷、神经炎症、神经退行性变以及认知功能均有所改善。其可能机制是腹膜透析可以减少血液中的Aβ,从而促进脑内的Aβ外流,改善脑内炎症微环境并增强小胶质细胞Aβ吞噬能力。外周清除最重要的基础是Aβ可以穿过血脑屏障(Blood Brain Barrier,BBB)。传统观点认为AD患者BBB转运功能受损,会减少脑内Aβ流出,促进外周Aβ内流。近期研究却提示AD病人以及APP/PS1小鼠的BBB功能相对完整。本研究中,我们首次探讨了血液Aβ的减少与脑内ISF中Aβ减少之间的动力学关系,研究结果支持APP/PS1小鼠BBB转运功能的完整性,也为外周途径清除脑内Aβ提供理论依据。我们进一步探讨了腹膜透析模型减少APP/PS1小鼠脑内Aβ的具体机制。通过计算,我们发现在治疗组中为期一个月腹膜透析直接清除的Aβ总量约为7.2ng。而10月龄的APP/PS1小鼠脑内的Aβ浓度约为1ng/mg,其大脑质量约为360mg,故可以计算出10月龄APP/PS1小鼠脑内AP总量约360ng。治疗组接受腹膜透析的APP/PS1小鼠脑内减少的Aβ为脑内AP总量的20.44%(约72ng)。因此,通过腹膜透析液直接清除的Aβ只占总Aβ减少量的10%,这提示在腹膜透析时还有其他的途径参与Aβ清除。通过免疫印迹实验发现在接受腹膜透析的APP/PS1小鼠LRP-1表达增加而RAGE表达减少,提示脑内Aβ向外周转运增强。另外,腹膜透析后其小胶质细胞Aβ吞噬的能力增强,促进了 Aβ的清除。此外,腹膜透析小鼠大网膜内Aβ沉积量明显高于对照组,提示大网膜也可能吸附部分Aβ。这些结果提示腹腔透析通过多种途径来实现对脑内Aβ的清除。当然,CKD患者与APP/PS1小鼠之间存在很多差异。课题组前期的工作已证实在CDK患者群体中,其血液中的Aβ水平显著高于正常对照组。这表明对肾脏功能受损的CDK患者而言,其外周Aβ清除功能也同样受损。而APP/PS1小鼠的肾功能虽然正常,但并不足以清除由于脑内Aβ过度产生引起的血液中急剧升高的Aβ。因此,CDK患者与APP/PS1小鼠之间都存在清除能力与Aβ水平之间不匹配的矛盾。因此,即使外周脏器功能没有受损,增强血液中Aβ清除能力也是AD防治的有效方法。另一个差异是,临床上CKD病人常用的透析方案为持续不卧床腹膜透析,全天都在进行透析。而本研究动物实验中APP/PS1小鼠每天仅接受两个小时的透析。这种短时间的透析便可以有效清除APP/PS1小鼠脑内Aβ,提示对AD患者而言,持续不卧床腹膜透析可能会取得更好的效果。而且,过去的尸检研究也支持透析疗法可以清除人体脑内的Aβ沉积。本研究为通过外周途径来清除脑内Aβ、恢复大脑内环境提供了概念验证性证据;也为通过外周途径来防治帕金森病、亨廷顿综合征以及肌萎缩侧索硬化等蛋白异常折叠的神经退行性疾病提供了新思路。结论腹腔透析能够通过清除血液Aβ、改善血脑屏障Aβ转运功能、增强脑内小胶质细胞Aβ吞噬能力等途径,清除脑内Aβ沉积、减轻AD相关病理、改善APP/PS1小鼠认知功能,提示腹腔透析是AD防治的潜在方法。
[Abstract]:Background and objective Alzheimer's disease (AD) is the most important type of dementia in the elderly. It is a neurodegenerative disease characterized by progressive memory impairment and cognitive dysfunction. The pathogenesis of.AD is complex. So far, the pathogenesis of the disease is not yet clear. The traditional view is that AD is the brain itself. The A beta plaques (A beta plaque) deposited by Amyloid-beta (A beta) in the brain are the most important pathological features of AD. The dominant mechanism of A beta cascade in the mainstream is that the main mechanism of AD is the excessive production or removal of A beta in the brain resulting in excessive deposition of AP in the brain, induced neuroinflammation, and excessive phosphorylation of Tau protein. It has been recognized in recent years that AD related pathology is not only limited to the brain. Research has found that A beta deposition is also found in the intestines, skin and heart, and may affect cardiac function. Our and other series of studies have also shown that chronic infection of the system, heart dysfunction, liver Systemic diseases such as renal insufficiency and chronic pulmonary infection are closely related to A beta metabolism or AD. It is suggested that the functional state of systemic organs may be involved in the development of AD. It is worth to discuss the pathogenesis and prevention strategy of AD from a systematic point of view. At present, there is no drug and method of.A beta to prevent or delay the development of AD, which is the current study on the prevention and control of AD. The most important target is a series of clinical trials on A beta production and removal in recent years, such as A beta active and passive immunotherapy, beta or gamma secretase inhibitors, but these tests have failed to achieve the expected effect. One of the important reasons for the failure of clinical trials is the severe side effects. In the clinical trials of immunotherapy, antibodies or As the drug enters the brain, it leads to the side effects of brain edema, micro bleeding, aseptic meningoencephalitis, and excitotoxicity of neurons, as well as the direct action of A beta in the brain. At the same time, the normal functioning of the brain needs a highly stable internal environment, and exogenous drugs enter the brain through the blood brain barrier, which can change the homeostasis of the brain. The potential risk of inducing adverse reactions. Avoiding drugs entering the brain and promoting A beta Exodus and scavenging through the removal of A beta in the brain may be a safer and more effective A beta clearance strategy. As a safe and effective AD treatment strategy, it is worthwhile to explore. Peritoneal dialysis (PD) is often used to remove blood and toxic substances in the brain. It is expected to be a new A beta peripheral clearance measure. Therefore, this subject is intended to explore the effectiveness of clearing blood A beta clearance in peritoneal dialysis by clinical and animal experiments. The effective prevention and treatment of AD is effective. The completion of these studies helps to understand the AD mechanism from a systematic perspective and to find effective preventive measures. Material and methods 1. collect blood samples from 30 patients with chronic renal disease (Kidney Disease, CKD) and Liquor Dialysisintraperitoneus samples for the first time of peritoneal dialysis, and to compare and compare three samples. The mouse peritoneal dialysis model was constructed with the internal A beta level.2.. The female C57 wild type mice (Wt mice) from the animal laboratory of Third Military Medical University were used as the blank control group. The APPswe/PS 1 dE9 (APP/PS 1) transgenic mice from the Jackson Laboratory of the United States were used as the peritoneal dialysis treatment group. The prevention group in the experimental group was set from 6 month old. From the beginning of dialysis to 7 month old termination, the treatment group was set to start from 9 month old to 10 month old to terminate, and the APP/PS1 mice who received catheterization but without dialysis were negative control group.3.. The microdialysis model.APP/PS1 mice and the Wt mice were the same as the same. 9 month old APP/PS1 mice were selected for microdialysis, and the above steps were set up to establish the abdomen. A microdialysis model was used in the microdialysis experiment. The microdialysis probe was implanted in the hippocampus and the microdialysis probe was implanted in the hippocampus. After 10 days, the wound healed and the needle core was inserted into the microdialysis process during the peritoneal dialysis. The negative control was taken from the.4. of Wt mice. After the excess of 6% chloral aldehyde intoxicated mice, the mice were opened to the thoracic cavity, the apical blood was taken, 0.85% of the blood. The normal saline was perfused through the apical puncture and cut open the right auricle. The brain was cut along the middle line to weigh the brain. The right hemisphere was fixed with 4% polyformaldehyde and used as immune staining. The left cerebral hemisphere was cut to about 100um and about Golgi staining. The rest of the liquid nitrogen was frozen at -80 C, used for biochemical analysis of.5.AD like pathological changes. .6E10,4G8 and Congo red staining were used to detect the ability of A beta plaque.6E10,4G8 to detect the phagocytosis of AP in microglia and.PS396 staining,.PS396 staining was used to detect Tau phosphorylation level.CD45 and GFAP for.6. ELISA detection of glial cell proliferation. Appropriate amount of brain tissue, TBS homogenate level intensity hyperacoustic, 100000 x centrifuge 1 hours, supernatant, For the soluble extract, the tube bottom tissue continued to add 200 mu L 2%SDS liquid, 10 grade intensity ultrasound, 100000 x g 4 C centrifuge for 1 hours, and take the supernatant for SDS extract. The tube bottom tissue continued to add 200 mu L 70%FA solution, 10 grade intensity ultrasonic treatment for 30 seconds, 100000 x G4 centigrade centrifuge 1 hours, and the supernatant was FA extract. ELISA kit was used to detect mice. Blood, after microdialysis, and the levels of A beta 40 and A beta 42 in the extracts of TBS, SDS and FA. The levels of inflammatory factors such as TNF- alpha, IFN- gamma, IL-1 beta, IL-6, IL-4 and IL-10 were also detected by ELISA method. The method was used to determine the total protein concentration of the sample. A suitable concentration of gel electrophoresis was used to carry out the immunoblotting experiment. The experiment mainly used anti A beta, APP-C, sAPP, pS199, pS396, PSD93, PSD95, synapsin-1, synaptophysin, LRP-1 and RAGE antibody detection target protein. Golgi staining was carried out, and the experimental methods were carried out strictly according to the instructions of the kit for.9. behavior and electrophysiological experiments. The prevention group and the APP/PS1 mice in the treatment group were all carried out by the Y- maze and the open field experiment. The treatment group was further examined by electrophysiological tests to detect the long history enhancement (Long-term Potentiation, LTP) in the hippocampus of APP/PS1 mice by peritoneal dialysis. Results 1. peritoneal dialysis reduced CKD patients and APP/PS1 mice blood A beta level.2. microdialysis experiment showed that peritoneal dialysis could reduce the A beta level of ISF in the brain of mice rapidly while reducing the A beta in the blood of mice, and the.3. in the mice blood and the A beta in ISF, in the preventive experiment and the treatment experiment, peritoneum permeation. The average level of A beta in the brain of APP/PS1 mice decreased the TNF- alpha in the brain of.4. peritoneal dialysis mice, IFN- gamma and IL-6 water decreased significantly, and the abnormal activation of microglia and astrocytes in the brain significantly decreased.5. peritoneal dialysis and increased the phagocytosis of microglia to A beta in the brain, and.6. peritoneal dialysis improved AP. P/PS1 mouse brain AD related pathology: hippocampal neuronal death decreased, tau protein phosphorylation level (pS199 and pS396) decreased, axon density increased, neuronal synapse spines increased.7. peritoneal dialysis to improve the cognitive function of APP/PS1 mice: the behavioral results of the treatment group, including the Y- maze (spontaneous alternating reaction and new arm recognition experiment) and the open field The experimental results have been significantly improved. Electrophysiological experiments suggest that peritoneal dialysis significantly improves the LTP. discussion in the hippocampus of APP/PS1 mice. We found that peritoneal dialysis can effectively reduce the levels of soluble A beta in the blood and ISF. After one month's dialysis, the A beta load, neuroinflammation, and neurodegenerative changes in the brain of APP/PS1 mice. The possible mechanism is that peritoneal dialysis can reduce the A beta in the blood, thus promote A beta Exodus in the brain, improve the microenvironment in the brain and enhance the phagocytosis of microglia A beta. The most important basis for peripheral clearance is that A beta can pass through the blood brain barrier (Blood Brain Barrier, BBB). The impaired BBB transport function can reduce the A beta outflow in the brain and promote the peripheral A beta flow. Recent studies have suggested that the BBB function of the AD patients and the APP/PS1 mice is relatively complete. In this study, we first explored the kinetic relationship between the decrease of A beta in the blood and the A beta reduction in the ISF in the brain. The results support the completion of BBB transport function of APP/PS1 mice. Integrality also provides a theoretical basis for the removal of A beta in the brain by peripheral pathways. We further explored the specific mechanism of A beta in the brain of APP/PS1 mice by peritoneal dialysis model. By calculation, we found that the total amount of A beta directly removed by peritoneal dialysis for one month in the treatment group was about 7.2ng. and the concentration of A beta in the brain of 10 month old APP/PS1 mice was approximately equal to that of the APP/PS1 mice. 1ng/mg, with a brain mass of about 360mg, can be calculated in the brain of 10 month old APP/PS1 mice that the total amount of AP in the brain of the 360ng. treatment group is 20.44% (about 72ng) in the brain AP in the APP/PS1 mice receiving peritoneal dialysis. Therefore, the A beta, which is directly removed by Liquor Dialysisintraperitoneus, is only 10% of the total A beta reduction, which suggests that in peritoneal dialysis. There were other ways to participate in A beta clearance. Through immunoblotting, the expression of LRP-1 in the APP/PS1 mice received peritoneal dialysis was increased and the expression of RAGE decreased, suggesting the enhancement of A beta transshipment in the brain. In addition, the ability of A beta phagocytosis in the microglia was enhanced after peritoneal dialysis, and the clearance of A beta was promoted. In addition, peritoneal dialysis mice omentum was also found. The amount of internal A beta was significantly higher than that of the control group, suggesting that the greater omentum may also adsorb partial A beta. These results suggest that peritoneal dialysis can achieve the clearance of A beta in the brain through a variety of pathways. Of course, there are many differences between the CKD patients and the APP/PS1 mice. The earlier work of the group has proved that the level of A beta in the blood is significant in the group of CDK patients. It was higher than the normal control group. This showed that the peripheral A beta scavenging function was also impaired in CDK patients with impaired renal function. While the renal function of APP/PS1 mice was normal, it was not sufficient to clear the A beta caused by the excessive production of A beta in the brain. Therefore, there was a clear clearance between CDK patients and APP/PS1 mice. A beta levels are inconsistent. Therefore, even if the peripheral viscera function is not damaged, the enhancement of A beta clearance in the blood is also an effective method for the prevention and treatment of AD. The other difference is that the commonly used dialysis scheme for the clinical CKD patients is continuous ambulatory peritoneal dialysis and is dialytic all day. In this study, the APP/PS1 mice were tested every day in this study. Only two hours of dialysis. This short time dialysis can effectively remove A beta in the brain of APP/PS1 mice, suggesting that continuous ambulatory peritoneal dialysis for AD patients may achieve better results. Moreover, past autopsy studies also support the removal of A beta in the brain of the human body. This study is through the peripheral pathway. It provides a conceptual proof for the removal of A beta in the brain and the restoration of the internal environment of the brain. It also provides new ideas for the prevention and treatment of abnormal foldable neurodegenerative diseases such as Parkinson's disease, Huntington's syndrome and amyotrophic lateral sclerosis. Conclusion peritoneal dialysis can improve the A beta transport of the blood brain barrier by removing the blood A beta. Function, enhancing the phagocytosis of A beta in brain microglia, clearing the A beta in the brain, alleviated AD related pathology and improving the cognitive function of APP/PS1 mice, suggesting that peritoneal dialysis is a potential method for the prevention and treatment of AD.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16

【参考文献】