6-羟多巴胺纹状体内注射制作大鼠帕金森病模型的研究

目的 为拓宽6-OHDA损毁多巴胺能神经元所制备大鼠帕金森病模型的应用范围,采用多位点纹状体内注入6-OHDA的途径来制备模型。方法 研究用SD大鼠,两个针道内四点定位注射,每点注射3μg／μ16-OHDA3μl。结果 术后两周出现缓慢旋转,4周旋转行为达到7转／分并保持稳定;形态学染色可见损毁1周后注射侧黑质酪氨酸羟化酶免疫组化阳性细胞减少20％,2周后减少38％,3～4周减少70％以上,6周后损伤趋缓。高效液相-电化学法活体检测纹状体内多巴胺的代谢产物3、4-二羟基苯乙酸(DOPAC)和高香草酸(HVA),发现注射侧和非注射侧相比含量分别下降98．33％和96．05％;组织匀浆检测损毁侧黑质多巴胺含量下降了73％以上,3、4-二羟基苯乙酸(DOPAC)含量下降60％。结论 纹状体内注射6-OHDA能够制备帕金森病大鼠模型。     

6-羟多巴胺脑内注射制备帕金森病大鼠模型的研究

目的　通过向大鼠脑内单侧、双点、间隔注射 6 OHDA ,建立PD动物模型。方法　取SD大鼠 4 0只 ,随机分为实验组 35只和对照组 5只。实验组大鼠右侧黑质致密部和内侧前脑束注射 6 OHDA ,两次注射间隔一周 ,对照组大鼠注射人工脑脊液 ,观察经阿朴吗啡诱导后大鼠的行为及黑质DA神经元形态学变化。结果　①实验组有 2 3只恒定左转鼠且旋转圈数 >2 10r 30min ,被认为是成功的PD模型 ,占 6 7 7% ;有 1只动物死亡 ,占 2 9%。②对PD大鼠模型的免疫组化研究发现 ,注射侧黑质区多巴胺神经元较健侧和对照组显著减少。结论　利用向脑内单侧、双点、间隔注射 6 OHDA制备PD大鼠模型 ,结果稳定可靠 ,动物死亡率低 ,为PD动物模型的建立提供了新方法。     

帕金森病模型大鼠行为学改变与黒质致密部多巴胺能神经元存活率之间的相关性

目的探讨线刀损毁内侧前脑束建立的帕金森病模型大鼠行为学改变与黒质致密部多巴胺能神经元存活率之间的相关性。方法采用可伸缩线刀切断大鼠内侧前脑束建立单侧损伤的帕金森病大鼠模型;皮下注射阿朴吗啡后测试大鼠的旋转行为;取中脑黑质切片进行酪氨酸羟化酶免疫组织化学染色,通过计数黑质致密部酪氨酸羟化酶阳性神经元的数目得到多巴胺能神经元存活率。结果模型组大鼠损伤侧酪氨酸羟化酶阳性神经元的数目明显下降,而由阿朴吗啡诱导的旋转行为明显增加。结论线刀损毁术后4周,帕金森病模型大鼠由阿朴吗啡诱导的旋转行为和黒质致密部多巴胺能神经元存活率之间存在着明显的负相关。     

多巴胺能神经元线粒体的氧化损伤与帕金森病

帕金森病(Parkinson’s disease,PD)的一个主要病理特征就是中脑黑质多巴胺能神经元的丧失,目前研究认为该病理变化与多种因素有关,包括蛋白质异常积聚、泛素蛋白酶体系统功能异常、神经炎症、线粒体损伤和氧化应激。在帕金森病人和动物模型中,中脑黑质有着明显的氧化改变。帕金森病的遗传和环境因素均会作用于线粒体,尤其对线粒体呼吸链复合体I有着抑制作用,造成线粒体损伤,产生活性氧(ROS)。活性氧的大量产生造成脂类、蛋白质和DNA的氧化,从而加剧多巴胺能神经元的线粒体和细胞损伤。多巴胺代谢过程中会产生活性氧,该自身代谢特点决定了多巴胺能神经元存在有较高的氧化应激,易受环境因素的影响。因而,线粒体的氧化损伤在帕金森病病理发生中起着重要作用。     

帕金森病大鼠中缝背核5-羟色胺能神经元放电频率和放电形式的变化

实验采用玻璃微电极细胞外记录法, 观察了帕金森病(Parkinson’s disease, PD)大鼠中缝背核(dorsal raphe nucleus, DRN)5- 羟色胺(5-hydroxytrypamine, 5-HT)能神经元电活动的变化。结果发现, 对照组和 PD 组大鼠 DRN 中 5-HT 能神经元的放电频率分别为(1.61 ±0.56) Hz 和(2.61 ±1.97) Hz, PD 组大鼠的放电频率显著高于对照组(P<0.05)。在对照组大鼠, 79% 的神经元呈现规则放电, 21% 为爆发式放电;在 PD 组大鼠,具有规则、不规则和爆发式放电的神经元比例分别为 36%、16% 和47%, 爆发式放电的 5-HT 能神经元比例明显高于对照组(P<0.05)。结果表明,帕金森病大鼠 DRN 中 5-HT 能神经元的放电频率增高, 且爆发式放电增多。     

早期帕金森病大鼠血清抗氧化能力降低

目的：研究早期帕金森病（PD）大鼠血清抗氧化能力的改变。方法：观察6-羟多巴胺（6-OHDA）早期PD大鼠多巴胺（DA）能神经元和血清抗氧化能力的异常改变。结果：早期PD动物DA能神经元的数量明显减少,血清抗氧化能力明显降低。结论：早期PD大鼠血清抗氧化能力降低,这可能与DA能神经元损伤有关。     

雌激素对6-羟基多巴胺损伤黑质多巴胺能神经元的保护作用

本研究以P50听觉诱发电位(P50 auditory evoked potential, P50)和酪氨酸羟化酶(tyrosine hydroxylase, TH)阳性细胞计数作为黑质功能和形态学指标,动态追踪研究雌激素对6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)损伤黑质多巴胺(dopamine, DA)能神经元的作用.将大鼠分为4组:(1)正常雌性大鼠对照组;(2)单纯帕金森氏病(Parkinson's disease, PD)模型组;(3)双侧去卵巢PD模型组;(4)去卵巢回补3d雌激素的PD模型组.在大鼠清醒和安静的生理状态下连续14d记录黑质的P50,并检测黑质TH 细胞数目的变化.结果显示:单纯PD模型大鼠黑质P50的T/C值较正常雌鼠降低40.60%(P<0.01),其损伤侧黑质TH 细胞数目减少64.74%(P<0.01);去卵巢PD模型大鼠黑质P50的T/C值较单纯PD模型大鼠进一步降低45.88%(P<0.01),同时其黑质TH 细胞数目值也进一步减少57.26%(P<0.01),表明急性缺乏生理水平性腺雌激素将增大6-OHDA损伤黑质DA能神经元的程度,同时使黑质的感觉门控(sensory gating, SG)功能明显受损;去卵巢后回补3d生理剂量雌激素,可明显改善大鼠黑质的SG功能,提高TH 细胞数量(与去卵巢PD模型大鼠比较,P<0.01),其黑质损伤程度与单纯PD模型大鼠相当.以上结果提示,生理水平的雌激素具有提高黑质DA能神经元对伤害性刺激耐受性的神经保护作用.缺乏性腺源性的雌激素时,及时给予生理剂量的雌激素可以减轻神经毒素6-OHDA对黑质DA能神经元结构和功能的损伤.     

毕赤酵母表达的neurturin对恒河猴帕金森氏病模型中黑质多巴胺能神经元的保护作用

帕金森氏病(PD)是由于多巴胺能神经元变性、坏死,导致黑质-纹状体系统的多巴胺含量下降而引起的一种神经系统退行性疾病,目前还没有一种很好的方法能使之治愈.Neurturin（NTN）能特异地作用于中脑多巴胺能神经元,对该类神经元具营养和保护作用.经静脉注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导恒河猴产生帕金森氏病模型,并在NTN治疗组,注射MPTP之前48 h脑室内注射重组毕赤酵母表达的人NTN 1 mg. 结果表明：模型组猴均逐渐出现了PD症状,而NTN治疗组猴,PD症状比较轻或不明显;荧光分光光度法测定MPTP模型组猴黑质、壳核和尾状核多巴胺（DA）、5-羟色胺（5-HT）和5-羟吲哚乙酸（5-HIAA）的含量结果与正常对照组相比均显著降低,NTN治疗组猴的黑质、壳核和尾状核中的DA、5-HT和5-HIAA与对照组相比无显著性差异,而与模型组相比,DA、5-HT和5-HIAA含量均明显增加;光镜检查MPTP模型组猴黑质神经元细胞明显脱失,而NTN治疗组猴黑质神经元细胞丢失不明显,与正常对照组猴无差别.上述结果表明,制备的重组人NTN在恒河猴体内能保护中脑黑质多巴胺能神经元不受MPTP的损伤,使其DA含量及多巴胺能神经元维持正常,在MPTP存在下没有发生PD症状.     

TET2在帕金森病细胞模型中的表达及机制研究

目的:迄今为止,帕金森病(PD)发生的分子机制尚未完全阐明,本研究旨在体外细胞模型中寻找PD新型表观遗传标志物,探索其发病机制。方法:本次研究使用的细胞为神经母细胞瘤细胞系SH-SY5Y。首先,我们用CCK-8检测细胞活力,选取合适浓度的MPP+构建PD细胞损伤模型。再用PBS和MPP+分别处理SH-SY5Y细胞,用RT-qPCR检测了几个甲基化酶与去甲基化酶DNMT1,DNMT3A,DNMT3B及TET1, TET2, TET3的mRNA的表达水平,并用蛋白印迹检测TET2蛋白水平,免疫荧光检测了TET2蛋白定位。进一步用慢病毒转染SH-SY5Y细胞敲低TET2后,检测细胞增殖。结果:本研究发现,MPP+对SH-SY5Y细胞增殖的抑制具有时间与浓度依赖性,我们最终选择2.5 mM MPP+作为后续的细胞处理浓度。与对照组相比,MPP+处理细胞TET2的mRNA及蛋白水平表达均增加,且蛋白进入细胞核增加;同时发现,敲低TET2表达可以延缓MPP+对SH-SY5Y细胞增殖的抑制作用。结论:在当前的研究中,我们报道了TET2蛋白可能是PD新型的表观遗传学标志物,提示我们将来也许可以使用TET2抑制剂来治疗PD,因此本研究有可能为PD提供新的治疗方向和靶点。     

溶酶体抑制剂对小鼠行为学与黑质多巴胺神经元的影响

目的初步探讨溶酶体抑制剂对小鼠行为学及多巴胺神经元功能的影响。方法于小鼠右侧中脑黑质（SN）区,立体定向微量注入溶酶体抑制剂和蛋白酶体抑制剂。观察阿朴吗啡诱导的小鼠旋转行为改变以及行为学变化;检测黑质区酪氨酸羟化酶（TH）阳性细胞数。结果阿朴吗啡未能诱导出小鼠旋转行为。蛋白酶体抑制剂组为10～15圈。多巴胺损害程度与溶酶体的剂量相关。磷酸氯喹25μmol/L时多巴胺神经元几乎没有损害作用;50μmol/L时局部区域多巴胺神经元有轻微损害;100μmol/L时损害明显;200μmol/L时黑质区注射局部神经元损害最严重,黑质注射区未见有神经元存在。1～4周呈现出较为明显逐渐恢复的特点。结论溶酶体功能抑制与多巴胺神经元凋亡相关,不同剂量溶酶体抑制剂在不同时间对多巴胺神经功能的损害不同。     

大白鼠黑质多巴胺细胞化学分化的顺序(英文)

本工作采用酪氨酸羟化酶(HT)免疫细胞化学技术结合电镜技术,研究了大白鼠黑质多巴胺细胞发育早期的分化和分布的特征。最早的TH阳性细胞首先于胚胎13天在中脑吻端的腹侧出现;随后出现的TH阳性细胞从尾端和背侧逐渐加入早先出现的TH细胞。在染色程度和细胞形态上,黑质区域内存在一个腹—背和外—内方向上的梯度,即位于腹侧和外侧的细胞染色较深、细胞体较大、核小、突起明显;而位于背侧和内侧的TH阳性细胞染色较淡、细胞体小、核大、突起不明显。最早的黑质——纹状体纤维由位于中脑黑质外侧部分的TH细胞首先发出。电镜下,发育早期的TH阳性细胞中都能见到粗面内质网。随着发育进程,TH阳性反应加深,粗面内质网和其它细胞器也相应增加。结合前人有关黑质细胞发生和迁移的放射自显影研究结果,本工作提示,黑质多巴胺细胞在停止迁移后开始化学分化,化学分化与细胞形态分化同步,并存在一定的时空先后顺序,这一顺序可能由神经发生的先后顺序决定。     

ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS

Abstract— In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE), further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl-β-methyl-choline (AcβMeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with AcβMeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide, inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS.     

脂多糖诱导黑质多巴胺能神经元变性中小胶质细胞的反应

目的探讨小胶质细胞活化规律与脂多糖(Lipopolysaccharide,LPS)诱导黑质多巴胺(DA)能神经元变性的关系。方法脑立体定位注射LPS入大鼠脑黑质后,采用特异性抗体0x．42标记不同时间点小胶质细胞的激活情况;酪氨酸羟化酶(tyrosine-hydroxylase,TH)免疫组织化学观察DA能神经元损害变化。结果小胶质细胞在LPS注入黑质6h后开始出现部分激活。12h大部分激活,为“灌木丛样”小胶质细胞;24h已完全激活,呈现“阿米巴样”,在随后的30d内,基本维持在此形态。而TH阳性细胞数在第3d开始出现下降,与对照组相比下降达45％,14d时下降至5～10％,至30d时几乎完全消失。结论小胶质细胞的激活先于DA能神经元变性,其激活介导的炎症反应在PD发病中具有重要的神经破坏作用。     

神经节苷脂GM1对帕金森模型小鼠黑质细胞凋亡及Bcl-2和Bax表达的影响

目的观察神经节苷脂GM1对帕金森模型小鼠黑质细胞凋亡及Bcl-2和Bax表达的影响,探讨其预防帕金森病的机制。方法成年C57-BL雄性小鼠分为正常对照组、模型组和神经节苷脂GMI治疗组。应用TUNEL法和免疫荧光组织化学染色技术观察各组小鼠黑质神经元凋亡及Bcl-2和Bax表达情况。结果TUNEL、Bcl-2和Bax阳性神经元主要位于黑质致密部。模型组黑质神经元凋亡数明显多于正常对照组,神经节苷脂GM1组黑质神经元凋亡数明显少于模型组。在模型组Bcl-2阳性神经元和Bax阳性神经元的数量均较正常对照组明显增多;在神经节苷脂GM1组Bcl-2阳性神经元的数量较模型组明显增多,但Bax阳性神经元的数量较模型组明显减少。结论神经节苷脂GM1可能通过增强黑质神经元内Bcl-2基因表达及抑制Bax基因表达的途径抑制黑质神经元的凋亡。     

臂旁核和延髓头端腹外侧区在黑质升压效应中的作用

本实验室观察到黑质具有升压效应。用L-谷氨酸钠微量注入黑质可使血压升高,此效应可被DA受体阻断剂氟哌啶醇(Halo)微量注入臂旁核加压区基本阻断。我们过去的工作证明延髓头端腹外侧区(RVL)及其内的α-受体中介臂旁核的加压效应,本实验将酚妥拉明注入RVL能明显衰减黑质的加压效应,而将Halo注入RVL加压区对黑质加压效应无明显影响。以上结果提示臂旁核-RVL(α-受体)加压系统参与黑质加压效应。     

THE SPONTANEOUS RELEASE OF ACETYLCHOLINESTERASE IN RAT SUBSTANTIA NIGRA IS ALTERED BY LOCAL CHANGES IN EXTRACELLULAR LEVELS OF DOPAMINE

Acetylcholinesterase release in the guinea-pig substantia nigra has been previously investigated ‘on-line’, using a sensitive chemiluminescent system. Since histological observations suggest that there is a difference in acetylcholinesterase distribution in the rat substantia nigra compared to that of the guinea-pig, the first aim of the present study was to use this chemiluminescent method to characterise acetylcholinesterase release in this brain region of the freely moving rat, and the second was explore the relationship between acetylcholinesterase release and dopamine systems in this region. Accordingly, acetylcholinesterase release in the rat substantia nigra was studied under basal conditions of spontaneous release and following the local administration of (a) elevated potassium ions (30, 45, 60 mM), (b) a stimulator of dopamine/acetylcholinesterase release—D-amphetamine (10⁻⁷, 10⁻⁶ and 10⁻⁵ M), (c) an inhibitor of dopamine uptake—GBR12909 (10⁻⁷, 10⁻⁶ and 10⁻⁵ M). Spontaneous release of acetylcholinesterase in this brain region of the rat appears to be comparable with that observed in the guinea-pig, despite the smaller number of acetylcholinesterase-containing neurones. Furthermore, not only elevated potassium ions, but

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-amphetamine as well as GBR12909, all produced significant increases in the percentage spontaneous release of acetylcholinesterase. Thus, the release of acetylcholinesterase in this region may be triggered by levels of dopamine outside of the neurone. Copyright © 1996 Elsevier Science Ltd     

中缝核提取液对体外培养的中脑黑质神经元存活和生长的影响

中脑黑质是中缝核５－ＨＴ神经元最早期的靶组织之一。为了分析神经元对其靶细胞的作用是受某种特异性蛋白或神经营养因子的影响,本文用中缝核提取液作用于体外培养的中脑黑质神经元,证明了该提取液能促进中脑黑质神经元的存活和生长,其活性部分是分子量大于３０ｋＤ的组份,经ＳＤＳ－聚丙烯酰胺梯度凝胶电泳,呈现一条主带,分子量在４３ｋＤ左右。     

DIFFERENTIATION OF DOPAMINERGIC AND NORADRENERGIC NEURONS IN RAT SPINAL CORD

Abstract— Norepinephrine (NE), dopamine (DM) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) content have been measured in different parts of rat spinal cord and cerebellum by a gas chromatographic mass spectrometric method. In cerebellum, which does not contain dopaminergic neurons, the ratio of NE to DA content was 47, whereas in parts of the spinal cord this ratio varied between 11 and 19. In the cord after desipramine (25 mg/kg, i.p.) plus 6-hydroxydopamine (6-HDA, 100/jg intracisternally), there was a significant depletion of DM but not of NE. Conversely, after benztropine (25 mg/kg, i.p.) plus 6-HDA there was a significant depletion of NE but not of DM. Chlorpromazine (10 mg/kg, i.p.) or clozapine (25 mg/kg, i.p.) caused a significant increase in spinal cord HVA concentration 1 h after treatment. Evidence is presented which suggests that the increased HVA measured in the cord did not originate in the brain. After electrolytic lesion of the locus coeruleus there was a significant reduction of NE but not of DM. Spinal cord DM and NE were depleted by reserpine in a dose-dependent manner, the threshold dose for DM depletion being less than that for NE depletion. Seven days after cord transection at T₁₀ spinal cord DM was significantly reduced in the lumbar region. These results suggest that dopaminergic neurons exist in rat spinal cord independently of noradrenergic neurons and that the DM is likely to be present in the terminals of descending axons.     

电针对帕金森病模型小鼠黑质铁染色细胞和铁蛋白表达的影响

目的探讨电针防治帕金森病的作用机制。方法采用1-甲基-4-苯基1,2,3,6四氢吡啶腹腔注射建立帕金森病小鼠模型,利用组织化学技术以及免疫组织化学技术观察电针对帕金森病小鼠脑黑质铁染色细胞和铁蛋白表达的影响。结果模型组1周、2周、4周小鼠黑质铁染色阳性细胞的数量较正常组明显增多,染色强度也明显增强（P〈0．001;P〈0．01;P〈0．001）,电针在3个存活期内均可明显减少帕金森病小鼠黑质铁阳性细胞的数量和染色强度（P〈0．001;P〈0．05;P〈0．001）;模型组1周、2周黑质部位铁蛋白的表达与正常组相比都有不同程度下降（P〈0．01;P〈0．001）,而电针组2周帕金森病小鼠黑质铁蛋白的表达与模型组相比显著增加（P〈0．01）。结论电针可以通过降低脑内铁的含量,同时增加铁蛋白的表达,从而提高帕金森病模型小鼠脑抗氧化能力,达到保护黑质多巴胺能神经元的目的。     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.