线粒体KATP通道开放剂对多巴胺能神经细胞的保护作用研究

目的研究线粒体KATP通道的开放对于鱼藤酮诱导的细胞凋亡的保护作用并初步探讨其机制。方法用神经生长因子(NGF)将PC12细胞诱导分化成多巴胺能神经元模型,经鱼藤酮和线粒体KATP通道的开放剂二氮嗪及选择性线粒体KATP通道拮抗剂5-羟葵酸(5-HD)处理,用台盼蓝染色和四甲基偶氮唑盐(MTT)法检测细胞活力,磷脂酰丝氨酸外翻法(Annexin V)检测细胞的凋亡,JC-1检测线粒体膜电位的变化。结果经鱼藤酮处理24h后PC12细胞突起结构消失,细胞体积变小,形态变圆,台盼蓝染色阳性细胞增多,细胞活力下降,可见An-nexin V阳性的早期凋亡细胞,凋亡率为31.1%±2.65%(P<0.01);同时加入二氮嗪能减少PC12细胞的凋亡,凋亡率为17.9%±0.71%(P<0.05);JC-1染色法证实二氮嗪可稳定线粒体膜电位。而同时加入5-HD的PC12细胞活力及线粒体膜电位与鱼藤酮处理组相比无变化。结论鱼藤酮可引起多巴胺神经元的凋亡,线粒体KATP通道的开放剂二氮嗪能够拮抗鱼藤酮的毒性作用,其机制可能是通过在线粒体膜电位降低时稳定线粒体膜电位而起到对多巴胺能细胞的保护作用。     

姜黄素通过清除小胶质细胞内活性氧类物质保护多巴胺能细胞

目的探讨小胶质细胞在多巴胺能细胞损伤中的作用,以及姜黄素通过抑制小胶质细胞反应保护多巴胺能细胞的机制。方法选用大鼠嗜铬细胞瘤株PC12细胞,利用鱼藤酮诱导其损伤建立帕金森病细胞模型,用姜黄素预处理4 h的小胶质细胞(BV-2)再经鱼藤酮处理4 h后的细胞上清作为条件培养液(microglia conditioned medium with curcumin,MCMC)处理PC12细胞,并设空白对照组。应用四甲基偶氮唑盐法(MTT法)检测细胞活力;DCFH-DA染色检测BV-2细胞内活性氧类物质(ROS)水平;磷脂结合蛋白(annexinⅤ)-碘化丙啶(PI)双染色流式细胞仪检测PC12细胞凋亡。结果 5nM鱼藤酮单独作用于PC12细胞,其存活率及凋亡率与空白组相比无明显差别(P>0.05);与对照组相比,姜黄素预处理的BV-2细胞,其ROS水平降低(P<0.01);受MCMC处理的PC12细胞,与对照组比较,细胞活力增加,细胞凋亡率降低(P<0.01)。结论鱼藤酮通过激活小胶质细胞产生ROS,损伤多巴胺能细胞。姜黄素通过抑制小胶质细胞反应,清除小胶质细胞内ROS,从而保护多巴胺能细胞。     

鱼藤酮诱导细胞内泛素化α-synuclein聚集选择性损伤多巴胺神经元

目的探讨鱼藤酮对多巴胺能神经元内泛素化-αsynuclein聚集的影响及其细胞损伤作用。方法应用鱼藤酮处理经NGF诱导的神经元样分化的PC12细胞株(多巴胺能神经元)与N2a细胞株(非多巴胺能神经元)4、8、16、24 h以及用利血平预处理PC12细胞4 h再加入鱼藤酮处理16 h;采用免疫荧光双标记方法在共聚焦显微镜下观察细胞内泛素化-αsynuclein聚集,以MTT法和流式细胞术分别检测PC12细胞活力及凋亡率。结果单用鱼藤酮处理16 h后,PC12细胞株与N2a细胞株相比较,免疫荧光双标记显示PC12细胞内泛素化-αsynu-clein发生明显聚集,并且鱼藤酮对PC12细胞株的作用具有时间依赖性,而N2a细胞内泛素化-αsynuclein聚集不明显。利血平耗竭PC12细胞内多巴胺后再经鱼藤酮处理,PC12细胞内泛素化-αsynuclein聚集不明显;经不同浓度鱼藤酮处理后,细胞活力呈剂量依赖性下降;与对照组相比,经20 nmol/L鱼藤酮处理4、16、24 h后细胞存活率分别为(81.6±12.3)%、(59.8±6.7)%和(52.2±7.4)%(P<0.01)。鱼藤酮处理后出现早期凋亡细胞,随着处理时间的延长细胞凋亡率逐渐上升(P<0.01)。结论鱼藤酮选择性作用于多巴胺能神经元,使细胞内泛素化-αsynuclein发生聚集,而且这种变化具有时间依赖性,最终导致细胞发生凋亡,然而鱼藤酮对非多巴胺能神经元作用不明显。同时,鱼藤酮诱导泛素化-αsynuclein发生聚集的作用与神经元的特性存在密切关系。     

鱼藤酮对多巴胺能神经元的神经毒性作用

目的　探讨鱼藤酮 (rotenone)对多巴胺能神经元的神经毒性作用的机制。方法　用神经生长因子 (NGF)将PC12细胞诱导分化成多巴胺能神经元的细胞模型 ,经不同浓度的鱼藤酮处理 ,观察细胞形态改变 ,四甲基偶氮唑盐 (MTT)法检测细胞活性及代谢状态 ,磷脂酰丝氨酸外翻法(Annexin V)检测细胞凋亡 ,流式细胞术检测细胞凋亡率 ,α synuclein、硫磺素T(thioflavinT)染色研究细胞内蛋白聚集情况。结果　经鱼藤酮处理 2 4h后PC12细胞突起样结构消失 ,细胞体积变小、形态变圆 ;随着鱼藤酮浓度或作用时间增加 ,细胞活性进一步下降 ,呈量效和时效依赖性。与对照组比较 ,细胞活力在浓度为 10nmol/L鱼藤酮作用 2 4h时即出现明显下降 ,吸光度A570 值为 0 4 15± 0 0 13(P <0 0 5 ) ;可见Annexin V呈阳性的早期凋亡细胞 ;凋亡率在 5nmol/L为 7 35 %± 0 5 2 % (P <0 0 5 ) ,在 10nmol/L为 13 30 %± 1 80 % (P <0 0 1) ;细胞内出现α synuclein和硫磺素T双标染色呈阳性的蛋白聚集。结论　鱼藤酮在体外对多巴胺能神经元有毒性作用 ,可诱导出现细胞凋亡并出现类包涵体 ,表明鱼藤酮可能通过影响α synuclein的代谢而在帕金森病发病机制中起作用。     

辛伐他汀减少三磷酸甘油醛脱氢酶的聚集及神经元凋亡

目的研究辛伐他汀对鱼藤酮诱导的三磷酸甘油醛脱氢酶(GAPDH)的聚集及对多巴胺能神经元凋亡的影响。方法体外培养PC12细胞,将细胞分为对照组、鱼藤酮处理组及鱼藤酮+辛伐他汀干预组;分别使用MTT试验了解药物干预对细胞活性的影响,使用生物化学方法检测细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力以及谷胱甘肽(GSH)含量,使用免疫组化了解细胞内GAPDH定位以及聚集的变化,采用流式细胞仪检测细胞凋亡情况。结果鱼藤酮处理能够诱导细胞内氧化应激的产生并使得GAPDH表达增加并聚集。辛伐他汀的处理能够降低细胞内MDA含量,提高SOD和GSH活性并减少GAPDH蛋白的表达及聚集,流式细胞仪凋亡检测也发现辛伐他汀的处理能够减少鱼藤酮诱导的细胞凋亡。结论辛伐他汀可能通过抗氧化作用而减少鱼藤酮诱导的GAPDH的聚集及细胞凋亡。     

丁基苯酞对帕金森病细胞模型保护作用的初步研究

目的探讨丁基苯酞(dl-3-n-Butylphthalide,NBP)对鱼藤酮诱导的帕金森病细胞模型的保护作用及其机制。方法分别使用终浓度为0.1、1、10、100μM NBP和溶剂二甲基亚砜(DMSO)预处理SH-SY5Y细胞24h后,加入终浓度为200nM的鱼藤酮处理24h建立多巴胺能细胞损伤模型,观察各组细胞形态,采用四甲基偶氮唑盐(MTT)比色法检测细胞活性,流式细胞术检测细胞凋亡率(Annexin V-FITC/PI)、线粒体膜电位(JC-1)、细胞内活性氧水平(DCFH-DA)。结果200nmol/L鱼藤酮处理SH-SY5Y细胞24h能够诱导细胞活性下降和细胞凋亡,NBP预处理后SH-SY5Y细胞存活率明显升高,细胞凋亡率降低,线粒体膜电位显著升高(P0.05),细胞内活性氧水平显著降低(P0.05),且随NBP浓度的增加对SH-SY5Y细胞的保护作用增强。结论NBP对鱼藤酮诱导的SH-SY5Y细胞损伤具有良好的保护作用,线粒体保护可能是其作用机制之一。     

利福平抑制鱼藤酮诱导PC12细胞的凋亡：通过减轻线粒体氧化应激可能实现？

背景：研究已表明利福平能在体内、外实验起重要的神经保护作用。尽管介导这一效应的确切机制尚不清楚,但一些研究提示利福平可能是通过拮抗线粒体功能障碍和氧化应激作用。 目的：探讨利福平对鱼藤酮诱导的分化PC12细胞线粒体氧化应激的保护作用和分析其可能机制。 设计、时间和地点：本实验是从2007年12月至2008年11月在中山大学附属第二医院神经科进行的随机重复测量的细胞实验。 材料：PC12细胞由中山大学中山医学院生理实验室馈赠。MTT购自美国MB chem.公司。鱼藤酮,利福平,罗丹明123,DCFH-DA,Hoechst 33342及Annexin V-FITC 凋亡检测试剂盒购自美国Sigma公司。还原型和氧化型谷胱甘肽测定试剂盒和蛋白定量试剂盒购自中国江苏碧云天生物技术研究所。 方法：PC12细胞经100 ng/ml 7S神经生长因子处理九天成分化细胞后培养于含有10%胎牛血清的DMEM/F12中,并孵育在含5% CO2的37 ℃培养箱中。隔天更换培养基。根据不同的处理条件分成六组：对照组（常规加培养基）、利福平组（300μmol/L利福平作用26h）、鱼藤酮组（2.5μmol/L鱼藤酮作用24h）和三组利福平预处理组（先分别给予100, 200 and 300 μmol/L利福平预处理2h,然后2.5μmol/L鱼藤酮再作用24h）。 主要结果测量：（1）MTT检测细胞活性;（2）Hoechst 33342核染和流式细胞仪检测细胞凋亡;（3）荧光显微镜和流式细胞仪测量经罗丹明123标记的线粒体膜电位;（4）流式细胞仪分析DCFH-DA标记的细胞内活性氧生成和酶标仪检测细胞内还原型谷胱甘肽。 结果：鱼藤酮诱导分化PC12细胞凋亡增加同时伴有细胞线粒体膜电位丢失、活性氧生成及谷胱甘肽耗竭;当细胞经过100, 200 and 300 μmol/L利福平预处理后,鱼藤酮诱导的这一线粒体功能障碍能够被利福平以剂量依赖方式所阻断。 结论：利福平预处理的分化PC12细胞能够通过减轻线粒体功能异常和氧化应激阻断鱼藤酮诱导的细胞凋亡。     

利福平对鱼藤酮诱导的分化PC12细胞线粒体损伤的保护作用

目的 探讨利福平(RFP)对鱼藤酮(Rot)诱导的分化PC12细胞活性、胞内活性氧(ROS)水平、线粒体膜电位(AWm)及凋亡的影响. 方法 利用Rot诱导的分化PC12细胞建立帕金森病(PD)细胞模型,应用不同浓度RFP(100、200、300 μmol/L)预先干预,分别采用MTT法检测PC12细胞活性、流式细胞仪检测胞内ROS生成量、荧光显微镜和流式细胞仪检测△ψm及凋亡的变化.结果 2.5 μmol/L Rot可使PC12细胞活性降低.ROS生成、△ψm去极化程度和细胞凋亡率增加;100、200、300 μmol/L RFP预处理对上述变化有抑制作用,且浓度越大,作用越明显. 结论 RFP可能通过稳定△ψm、降低细胞内ROS生成来对抗Rot对分化PC12细胞的损伤,且这种作用呈浓度依赖性.     

姜黄素抑制小胶质细胞NADPH氧化酶保护多巴胺能细胞的机制研究

目的：探讨小胶质细胞在多巴胺能细胞损伤中的作用,以及姜黄素通过抑制小胶质细胞反应保护多巴胺能细胞的机制。方法选用大鼠嗜铬细胞瘤株PC12细胞,用鱼藤酮诱导其损伤建立帕金森病细胞模型;用姜黄素预处理4 h的BV‐2细胞再经鱼藤酮处理4 h后的细胞上清液作为条件培养液（microglia conditioned medium with curcumin ,MCMC）,处理PC12细胞。应用四甲基偶氮唑盐法（MTT法）检测细胞活力;DCFH‐DA染色检测BV‐2细胞内活性氧类物质（ROS）的水平;Western blotting法检测NADPH氧化酶P47‐phox亚基在BV‐2细胞膜上的表达。结果与对照组相比,经姜黄素预处理的BV‐2细胞ROS水平降低（P＜0.01）;受MCMC处理的PC12细胞活力增加（P＜0.01）。姜黄素预处理使结合到BV‐2细胞膜的NADPH氧化酶P47‐phox亚基明显降低（P＜0.05）。结论鱼藤酮通过激活小胶质细胞内NADPH氧化酶产生ROS ,损伤多巴胺能细胞。姜黄素能够抑制小胶质细胞内NADPH氧化酶的激活,减少ROS的产生,进而保护多巴胺能细胞。     

溶酶体自噬途径降解α-synuclein蛋白作用研究

目的 探讨α-synuclein蛋白细胞内溶酶体途径降解机制.方法 用神经生长因子NGF诱导分化PC12细胞作为研究多巴胺能神经元的细胞载体,应用鱼藤酮处理PC12细胞建立α-synuclein蛋白细胞模型.使用溶酶体途径降解抑制剂E64处理神经元样分化的PC12细胞,应用免疫荧光双标方法观察PC12细胞内硫黄素S、α-synuclein蛋白阳性聚集包涵体形成情况,比较各组的差异.结果 用E64处理鱼藤酮预处理过的PC12细胞后α-synuclein蛋白聚集且较多包涵体形成(15.36±0.85)%,与对照组相比差异有统计学意义(P＜0.05).结论 溶酶体自噬途径可能在α-synuclein蛋白降解、聚集和多巴胺神经元死亡过程中发挥重要作用.     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.