雷帕霉素抗神经退行性病研究进展

雷帕霉素作为一种传统的抗真菌药物，具有免疫抑制剂和抗肿瘤的作用。值得注意的是，它通过抑制哺乳动物雷帕霉素靶标蛋白mTOR、受体FKBP、VMP1，促进自噬，抑制了帕金森病、亨廷顿病等神经退行性变的病理过程，从而有望成为一种新的抗神经退行病药物。本文对其新的研究进展予以综述。     

雷帕霉素激活自噬改善嘌呤霉素氨基核苷诱导的足细胞损伤

目的:通过观察雷帕霉素对PAN 诱导的足细胞损伤及自噬相关蛋白表达的影响,探讨自噬在雷帕霉素保护PAN 诱导的足细胞损伤中的作用及可能机制。方法:构建PAN 诱导的足细胞损伤模型,将足细胞分成对照组(Control 组),PAN 组(加入50 μg/ ml PAN),雷帕霉素组(RAP 组:分别加入100、200、300 ng/ ml 雷帕霉素),PAN+雷帕霉素组(PAN+RAP组:细胞在用含PAN 的培养液培养前1 h,分别用100、200、300 ng/ ml 雷帕霉素进行预处理1 h)。采用Annexin V/ PI 双染法检测细胞凋亡,透射电镜观察自噬小体,Western blot 检测LC3、p62、4EBP1、P70S6K、mTOR 蛋白表达。结果:与对照组比较,PAN组足细胞凋亡增加,自噬体减少,LC3域蛋白表达下调,p62 上调,mTOR、4EBP1、P70S6K 磷酸化水平上调;与PAN 组比较,PAN+RAP 组足细胞凋亡率下降,自噬体增加,LC3域蛋白表达上调,p62 下调,mTOR、4EBP1、P70S6K 磷酸化水平下调。结论:PAN 可以抑制足细胞自噬,促进足细胞凋亡;雷帕霉素可通过激活自噬改善PAN 诱导的足细胞损伤,这种作用可能与雷帕霉素抑制mTOR/4EBP1、P70S6K 信号通路有关。     

自噬与间充质干细胞治疗神经退行性病的相互作用

背景：神经元的自噬功能紊乱和异常蛋白质聚集是神经退行性疾病的主要病理改变,间充质干细胞与自噬的联系及其相互作用代表一种治疗神经退行性疾病可能的机制。目的：围绕自噬与间充质干细胞治疗神经退行性疾病及其相互作用的研究进展进行综述,旨在为治疗神经退行性疾病提供理论依据和新思路。方法：在PubMed和中国知网数据库以“autophagy,neurodegenerative diseases,mesenchymal stem cells,Parkinson’s disease,Alzheimer’s disease”为英文检索词,以“自噬,神经退行性疾病,间充质干细胞,帕金森病,阿尔茨海默病”为中文检索词,检索相关文献,最终纳入59篇文献进行综述。结果与结论：(1)自噬平衡有益于维持中枢神经系统内外环境的稳定性以及控制神经退行性疾病的病程进展;(2)自噬作为一种维持细胞更新和平衡状态的动态循环机制,可影响间充质干细胞的迁移、存活、分化、抗凋亡和免疫调控等生物学功能,同时优化其对疾病的治疗疗效;(3)间充质干细胞是一类重要的神经保护剂,可通过调节细胞自噬水平高低,以此减轻神经退行性疾病的病理学特征...     

神经干细胞自噬与自噬机制研究现状

<正>自噬(autophagy)作为细胞内物质代谢的重要方式,是亚细胞膜内的结构发生动态变化,也是溶酶体介导细胞内蛋白质和细胞器降解的正常新陈代谢过程。细胞死亡包括坏死、凋亡和自噬三种形式。在细胞平衡合成和分解代谢过程中,通过自噬保持细胞内环境的稳定,使细胞自我更新,保持活力[1-4]。自噬不足则细胞衰老,过度则出现程序性细胞死亡     

长春新碱诱导的自噬性细胞凋亡对线粒体膜电位的影响

目的：了解长春新碱（VCR）诱导的自噬性细胞凋亡是否与线粒体跨膜电位（△Ψm）改变有关及其可能的机制。材料与方法：以正常人的肝细胞系为体外模型,应用碘化丙啶（PI）／Rhodamine123(Rh123)双重染色检测△Ψm,通过亚GI期细胞和透射电镜鉴定细胞凋亡。结果与结论：VCR可明显诱导线粒体△Ψm下降,也可以诱导L－02细胞自噬性凋亡。线粒体△Ψm下降可能是VCR诱导自噬性细胞凋亡的关键环节。     

内质网应激诱导的自噬对肝细胞凋亡的影响

目的:观察二硫苏糖醇(DTT)诱导人正常肝细胞LO2发生内质网应激的过程中自噬相关指标表达的变化及其对细胞凋亡的影响。方法:采用2.0 mmol/L DTT处理LO2细胞0、6、12和24 h,诱导细胞发生内质网应激;real-time PCR及Western blot检测葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、活化转录因子4(ATF4)、C/EBP同源蛋白(CHOP)、自噬相关基因12(Atg12)、自噬相关基因5(Atg5)和微管相关蛋白1轻链3(LC3) mRNA及蛋白水平的变化;流式细胞术检测细胞凋亡情况;透射电镜观察自噬小体的产生情况。采用400 nmol/L的雷帕霉素预处理LO2细胞1 h,再给予2.0 mmol/L的DTT处理24 h,观察雷帕霉素预处理对细胞凋亡的影响。结果:2.0 mmol/L DTT处理LO2细胞6、12和24 h后,细胞中GRP78、PERK、ATF4、CHOP、Atg12、Atg5和LC3的mRNA及蛋白水平较0 h组显著上调(P 0.05);同时LC3Ⅱ/LC3Ⅰ也较0 h组显著增高(P 0.05);透射电镜检测发现,DTT处理6、12和24 h后,细胞中均可见自噬小体的产生;流式细胞术检测发现,DTT处理细胞6、12和24 h后,细胞凋亡较0 h组显著增加;而经自噬诱导剂雷帕霉素预处理后,DTT诱导的细胞凋亡显著下降(P0.05)。结论:内质网应激可诱导自噬的发生,而雷帕霉素预处理可在一定程度上减轻内质网应激诱导的LO2细胞凋亡。     

自噬通过抑制凋亡促进体外饥饿状态下脂肪细胞存活

 目的: 观察体外饥饿状态下脂肪细胞的自噬变化,研究自噬在维持体外饥饿状态下脂肪细胞存活中的作用。方法: 将小鼠3T3-L1细胞诱导成为脂肪细胞后,雷帕霉素(rapamycin,RAP)预处理脂肪细胞,在缺糖缺氧(oxygen-glucose deprivation,OGD)模拟的饥饿环境中孵育细胞。应用Western blotting及透射电镜法检测细胞自噬变化,用TUNEL染色及Western blotting检测脂肪细胞凋亡。结果: OGD条件下脂肪细胞的自噬水平明显升高,RAP预处理进一步上调了OGD条件下的细胞自噬。与对照组相比,OGD组细胞cleaved caspase-3的水平及细胞凋亡率明显升高(P<0.01)。RAP预处理降低了OGD 条件下cleaved caspase-3的水平,并明显降低了细胞凋亡率(P<0.05)。结论: 自噬在饥饿状态下脂肪细胞存活中发挥保护性作用,提高自噬水平有助于降低饥饿状态下脂肪细胞的凋亡水平。     

线粒体自噬在器官纤维化疾病中作用的研究进展

线粒体自噬是一种选择性降解细胞中损伤或多余线粒体的特异性自噬现象,使细胞在应激损伤时能够维持线粒体数量和质量的稳定,从而维持细胞的正常表型和功能。其分子机制途径较为复杂,主要涉及PINK1/Parkin途径、NIX和BNIP3、FUNDC1等。线粒体自噬的异常与多种疾病密切相关,而调节不同阶段线粒体自噬分子机制在疾病发展中的作用已被重视。现就近年线粒体自噬在多种器官纤维化病变中的研究进展综述如下。     

神经退行性疾病中的自噬与再生研究进展

神经退行性疾病是由于神经元纤维的缠结和神经元的丢失所致,而产生功能障碍的现象,神经干细胞(NSCs)的损伤和生成减少也会引起神经退行性疾病的产生。自噬可以降解受损的细胞和细胞器,自噬在NSCs的增殖和分化中起着重要作用。mTOR和Notch都可以通过调节自噬来控制NSCs的生成,抑制mTOR信号通过干扰自噬作用于年龄相关的神经退行性疾病,自噬的改变可以影响Notch信号对NSCs分化的影响,本文归纳总结了自噬与再生在神经退行性疾病中作用的研究进展。     

Stem cell therapies in age-related neurodegenerative diseases and stroke

Aging, a complex process associated with various structural, functional and metabolic changes in the brain, is an important risk factor for neurodegenerative diseases and stroke. These diseases share similar neuropathological changes, such as the formation of misfolded proteins, oxidative stress, loss of neurons and synapses, dysfunction of the neurovascular unit (NVU), reduction of self-repair capacity, and motor and/or cognitive deficiencies. In addition to gray matter dysfunction, the plasticity and repair capacity of white matter also decrease with aging and contribute to neurodegenerative diseases. Aging not only renders patients more susceptible to these disorders, but also attenuates their self-repair capabilities. In addition, low drug responsiveness and intolerable side effects are major challenges in the prevention and treatment of senile diseases. Thus, stem cell therapies—characterized by cellular plasticity and the ability to self-renew—may be a promising strategy for aging-related brain disorders. Here, we review the common pathophysiological changes, treatments, and the promises and limitations of stem cell therapies in age-related neurodegenerative diseases and stroke.     

Aging: An important factor for the pathogenesis of neurodegenerative diseases

Aging is a natural process that is defined as a progressive deterioration of biological functions after the organism has attained its maximal reproductive competence. Aging leads to the accumulation of disabilities and diseases that limit normal body functions and is a major risk factor for neurodegenerative diseases. Many neurodegenerative diseases share oxidative stress and nitrosative stress as common terminal processes. According to free radical theory of aging, an elevation in reactive oxygen species (ROS) and reactive nitrogen species (RNS) damages neural membranes and induces oxidative and nitrosative stress. The increase in oxidative and nitrosative stress is accompanied by the concomitant decline in cognitive and motor performance in the elderly population, even in the absence of neurodegenerative diseases. Markedly increased rates of oxidative and nitrosative stress are the major factors associated with the pathogenesis of neurodegenerative diseases. Diet is a key environmental factor that affects the incidence of chronic neurodegenerative diseases. Dietary supplementation with polyphenols, resveratrol, ginkgo biloba, curcumin, ferulic acid, carotenoids, flavonoids, and n-3 fatty acids exerts beneficial effects not only through the scavenging of free radicals, but also by modulating signal transduction, gene expression, and restoring optimal neuronal communication.     

雷帕霉素对顺铂作用下人肺腺癌A549及耐药A549/DDP细胞增殖、侵袭、黏附及自噬凋亡的影响

 目的:研究雷帕霉素（Rap）对顺铂（DDP）作用下人肺腺癌A549及耐药A549/DDP 细胞增殖、迁移、黏附及其自噬凋亡的影响。方法: 培养人肺腺癌A549及耐药A549/DDP细胞株,利用MTT方法分别检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞增殖抑制率的影响;Transwell方法检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;黏附实验检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;流式细胞术检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞凋亡的影响;Western blotting检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞自噬标志蛋白beclin-1和LC3表达的影响。 结果: 与Rap或DDP单独作用组相比,Rap和DDP联合作用能够同时显著抑制人肺腺癌A549及耐药A549/DDP细胞增殖、体外侵袭能力及细胞黏附能力,并能够促进细胞凋亡和自噬标志蛋白beclin-1和LC3的表达（均P＜0.05）。结论: Rap能够通过促进细胞自噬而增强DDP的作用,进而抑制人肺腺癌A549及耐药A549/DDP细胞的增殖、侵袭、黏附并促进细胞凋亡作用,具有协同作用。     

Mitochondrial function and dynamics in neural stem cells and neurogenesis: Implications for neurodegenerative diseases

Mitochondria have been largely described as the powerhouse of the cell and recent findings demonstrate that this organelle is fundamental for neurogenesis. The mechanisms underlying neural stem cells (NSCs) maintenance and differentiation are highly regulated by both intrinsic and extrinsic factors. Mitochondrial-mediated switch from glycolysis to oxidative phosphorylation, accompanied by mitochondrial remodeling and dynamics are vital to NSCs fate. Deregulation of mitochondrial proteins, mitochondrial DNA, function, fission/fusion and metabolism underly several neurodegenerative diseases; data show that these impairments are already present in early developmental stages and NSC fate decisions. However, little is known about mitochondrial role in neurogenesis. In this Review, we describe the recent evidence covering mitochondrial role in neurogenesis, its impact in selected neurodegenerative diseases, for which aging is the major risk factor, and the recent advances in stem cell-based therapies that may alleviate neurodegenerative disorders-related neuronal deregulation through improvement of mitochondrial function and dynamics.     

肿瘤坏死因子相关凋亡诱导配体参与雷帕霉素诱导血管内皮细胞功能损伤的体外研究

目的: 探讨雷帕霉素对内皮细胞凋亡和增殖、迁移能力的影响,以及肿瘤坏死因子相关凋亡诱导配体(TRAIL)表达水平的变化。方法: 用浓度为0、1、10、100 μg/L 的雷帕霉素孵育内皮细胞24 h,应用CCK8法检测血管内皮细胞的增殖能力,Transwell小室和划痕试验检测细胞迁移能力,DAPI染色观察凋亡细胞核形态改变,Western blotting法检测caspase-3活性以显示血管内皮细胞的凋亡,并用 Western blotting检测TRAIL在凋亡的内皮细胞中的表达。结果: 雷帕霉素(1-100 μg/L)能诱导血管内皮细胞凋亡并抑制其迁移能力(P<0.01)。除雷帕霉素1 μg/L外, 10 μg/L和100 μg/L雷帕霉素均能抑制内皮细胞增殖能力(P<0.01),同时雷帕霉素(10-100 μg/L)使TRAIL蛋白表达增加,两者作用均呈浓度依赖性(P<0.01)。结论: 雷帕霉素能诱导内皮细胞发生凋亡并抑制其增殖和迁移能力。TRAIL表达上调与雷帕霉素诱导血管内皮细胞损伤有一定的相关性。     

Akt激酶抑制剂MK-2206诱导U2OS人骨肉瘤细胞凋亡与自噬

 目的：研究Akt抑制剂MK-2206对U2OS细胞凋亡和自噬的影响。方法：采用MTT法检测MK-2206对U2OS细胞活力的影响,DNA片段末端标记试剂盒检测细胞凋亡的变化,免疫印迹法检测细胞内蛋白的表达,用LC3-II的表达量用来确定细胞的自噬水平。结果：MK-2206剂量依赖性地降低了U2OS细胞的活力;MK-2206能够促进caspase-9、caspase-3和PARP的活化切割而诱导U2OS细胞发生凋亡;MK-2206给药后可促进细胞内LC3-II的表达,氯喹阻断自噬后明显增强了MK-2206对U2OS细胞活力的抑制作用。结论：Akt 抑制剂MK-2206能够诱导U2OS细胞发生凋亡和自噬;抑制自噬可促进MK-2206对U2OS细胞的毒性。     

Heart rate variability in relation to cognition and behavior in neurodegenerative diseases: A systematic review and meta-analysis

Neurodegenerative diseases, which frequently present with neuropsychiatric symptoms related to prefrontal cortical dysfunction, can alter the integrity of the neural networks involved in central autonomic nervous system regulation, which is proposed to be indexed by heart rate variability (HRV). We systematically reviewed the characteristics, methodology and outcomes of 27 studies of HRV in relation to measures of cognition and behavior in neurodegenerative conditions, and assessed the strength of this relationship, cross-sectionally, across 18 studies. A significant, moderate effect was observed (r = 0.25), such that higher HRV was related to better cognitive and behavioral scores, which was not influenced by mean age or cognitive status. There was no evidence of small-study effects but we could not rule out publication bias, and other factors may have contributed to heterogeneity between studies. Our findings support the proposal that HRV may be a marker of self-regulatory processes in neurodegenerative conditions, and further research on this association is needed in relation to neuropsychiatric symptoms and alongside neuroimaging methods.     

骨髓间充质干细胞的组织亲和性与脑片移植

目的 检测骨髓间充质干细胞的组织亲和性、时间亲和性以及移植治疗神经元退行性疾病的可行性。 方法 应用组织细胞共培养和免疫细胞化学的方法观察骨髓间充质干细胞在不同组织的微环境下的黏附和向神经细胞的分化情况。 结果 与肝片、肺片相比,骨髓间充质干细胞在海马脑片上的黏附力最强;骨髓间充质干细胞在生后7d（P7）小鼠脑片上的黏附力最强,并随着年龄的增长呈逐渐递减的趋势;骨髓间充质干细胞在正常野生型小鼠和阿尔茨海默病小鼠脑片上都能向神经细胞分化,并且两者无显著差异。 结论 骨髓间充质干细胞更适宜用于脑部疾病的治疗,并且年龄越小移植效果越好。     

Activation of 70-kDa S6 kinase,induced by the cytokines interleukin-3 and erythropoietin and inhibited by rapamycin,is not an absolute requirement for cell proliferation

The cytokines interleukin (IL)-3 and erythropoietin (EPO) are critical regulators of the proliferation and differentiation of cells of the hematopoietic system, but their intracellular mechanisms of action are not fully understood. Binding of IL-3 to the IL-3 receptor (IL-3R) and binding of EPO to the EPOR both induce changes in intracellular tyrosine and serine/threonine phosphorylation; the phosphorylation of a number of polypeptides appears to be a shared response upon cytokine stimulation. We have previously shown that binding of IL-2 to the IL-2R activates the 70-kDa (p70) S6 kinase, a serine/threonine kinase whose activity is regulated by serine/threonine phosphorylation; the immunosuppressant rapamycin inhibits IL-2-dependent proliferation and IL-2-triggered activation of p70 S6 kinase. We, therefore, sought to examine whether induction of p70 S6 kinase activity is a conserved response upon cytokine triggering, and whether this activity is essential for cell proliferation. Proliferation of the IL-3-dependent pro-B cell line Ba/F3 transfected with the EPOR (Ba/F3-EPOR) can be supported by either IL-3 or EPO. In this cell line, both IL-3 and EPO induced p70 S6 kinase activity; rapamycin inhibited both the IL-3 and EPO-induced activation of the 70-kDa S6 kinase as well as cellular proliferation. Thus, p70 S6 kinase activation appears to be a common intermediate triggered by the stimulation of IL-3, EPO, and IL-2 receptors. The Friend spleen focus-forming virus gp55 renders the EPOR constitutively active, and confers growth factor independence on cells expressing EPOR. Ba/F3-EPOR cotransfected with gp55 (Ba/F3-EpoRgp55) and the erythroleukemia cell line MEL, which also expresses both the EPOR and gp55, were analyzed. Rapamycin inhibited the activation of p70 S6 kinase in both cell lines. However, rapamycin inhibited proliferation of Ba/F3-EpoRgp55 but not of MEL cells despite inhibition of p70 S6 kinase activity in both cells. Thus, p70 S6 kinase activation is not an absolute requirement for cell proliferation. These results are discussed in relation to the role of the activation of the 70-kDa S6 kinase activation pathway in the regulation of cell cycle progression.