帕金森病小鼠黑质多巴胺能神经元凋亡诱导因子核移位的研究

目的探讨帕金森病(PD)小鼠模型黑质凋亡诱导因子(AIF)的核移位情况及其与多巴胺(DA)能神经元损伤之间的关系。方法采用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)制备PD小鼠模型,2h、24h、72h后取中脑组织进行酪氨酸羟化酶免疫染色观察黑质DA能神经元的损害情况,AIF免疫组化染色和Western blot方法检测AIF的核移位情况。结果与对照组比较,PD组小鼠黑质DA能神经元数量随时间呈递减趋势,并出现AIF核移位,2h时达高峰,之后随时间呈下降趋势。结论AIF核移位是PD小鼠黑质DA能神经元损伤的早期指标,在PD的发病过程中发挥重要作用。     

p38MAPK抑制剂对MPTP模型小鼠黑质多巴胺能神经元的保护作用

目的:研究SB239063在1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)所致帕金森病(PD)模型小鼠中抑制p38丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)活化对多巴胺(DA)能神经元的保护作用。方法:雄性C57BL/6N小鼠随机分为MPTP(30 mg/kg)模型组;SB239063(5 mg/kg)抑制剂组;SB239063(15 mg/kg)抑制剂组;SB239063(25 mg/kg)抑制剂组。抑制剂组于每次注射MPTP前3 h腹腔注射SB239063;对照组注射与模型组和抑制剂组等量生理盐水和DMSO。采用免疫组织化学和免疫蛋白印迹法观察各组小鼠黑质酪氨酸羟化酶(TH)和磷酸化p38蛋白激酶(p-p38 protein kinase,p-p38MAPK)之间表达变化的关系。结果:模型组小鼠黒质区p38MAPK显著活化,同时伴有TH阳性神经元明显丢失;SB239063 15 mg/kg与25 mg/kg组均可明显减少TH神经元丢失,而5 mg/kg组无显著影响;免疫荧光双标记结果显示p38MAPK与TH阳性神经元存在共表达。结论:p38MAPK对PD模型小鼠中脑黑质多巴胺能神经元丢失可能有重要调控作用,SB239063对多巴胺神经元具有一定的神经保护作用。     

环加氧酶-2对帕金森病模型小鼠黑质多巴胺能神经元的影响

目的　观察COX 2对帕金森病小鼠黑质多巴胺能神经元的影响。方法　选用C5 7BL种系环加氧酶 2 (Cyclooxygenase 2 ,COX 2 )缺陷小鼠 ,腹腔注射 1 甲基 4 苯基 1,2 ,3,6 四氢吡啶 (MPTP)制备帕金森病小鼠模型 ,用免疫组织化学方法。结果　行为学及免疫组织化学观察显示 ,野生型帕金森病小鼠的死亡率明显高于COX 2缺陷杂合子帕金森病小鼠 (P <0 0 1) ;野生型帕金森病小鼠黑质致密部酪氨酸羟化酶 (Ty rosineHydroxylase ,TH)免疫反应阳性神经元数目较杂合子帕金森病小鼠明显减少 (P <0 0 1)。结论　COX 2很可能与帕金森病时黑质多巴胺能神经元的损伤有关     

慢性帕金森病模型小鼠的行为学表现和多巴胺能神经细胞及转运体的数量变化

目的:研究慢性帕金森病模型小鼠行为学、黑质致密部多巴胺神经细胞和纹状体多巴胺转运体(DAT)的变化。方法:利用MPTP和二丙苯磺胺联合注射小鼠5周(每周2次,共10次)制作帕金森病小鼠慢性模型,通过转棒法检测小鼠行为学表现,免疫组织化学方法测定黑质内多巴胺能神经细胞的变化,放射自显影技术测定纹状体内DAT的密度。结果:帕金森病小鼠在转棒试验中行为发生明显改变,模型组行为学得分明显低于对照组(895±238 vs 1202±252,P0.001),酪氨酸羟化酶(TH)阳性细胞数也明显低于对照组(10.4±6.5 vs22.4±8.0,P0.001)。模型组动物纹状体DAT放射自显影灰度值明显低于对照组(0.2123±0.0316 vs 0.3034±0.0588,P0.001)。线性回归表明,模型组小鼠黑质致密部TH阳性细胞数及纹状体内DAT密度与对应行为学得分呈明显正相关(r=0.6354,P0.01)。结论:MPTP和二丙苯磺胺所致的帕金森病小鼠,在行为学、纹状体DAT密度和黑质致密部多巴胺神经细胞量上均有明显改变,提示这种小鼠模型可用于研究帕金森病的发病过程和药物治疗。     

银杏叶提取物对胚胎大鼠中脑原代细胞及多巴胺能神经元活性的影响*

背景：银杏叶提取物具有清除自由基和过氧化脂质,对抗兴奋性神经递质释放等多种药理活性。 目的：观察银杏叶提取物EGB761对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinum, MPP+)及左旋精氨酸(L-arginine, L-Arg)干预后的中脑原代细胞及多巴胺能神经元是否具有保护作用。 方法：分离培养SD胚胎大鼠中脑神经细胞,分组培养：空白对照组、MPP+组、L-Arg组、MPP++L-Arg组、MPP++EGB761组、MPP++EGB761+L-Arg组。 结果与结论：①细胞A值：MPP+组、MPP++L-Arg组显著低于空白对照组、L-Arg组、MPP++EGB761组及MPP++EGB761+L-Arg组(P < 0.05),MPP++L-Arg组低于MPP+组(P < 0.05),MPP++EGB761+L-Arg组低于MPP++EGB761组(P < 0.05)。②酪氨酸羟化酶阳性细胞数：MPP+组、MPP++L-Arg组显著低于空白对照组、L-Arg组、MPP++EGB761组及MPP++EGB761+L-Arg组(P < 0.05),MPP++L-Arg组低于MPP+组(P < 0.05),MPP++EGB761+L-Arg组低于MPP++EGB761组(P < 0.05)。③细胞一氧化氮水平：MPP+组、MPP++L-Arg组显著高于空白对照组、L-Arg组、MPP++EGB761组及MPP++EGB761+L-Arg组(P < 0.05),MPP++L-Arg组高于MPP+组(P < 0.05),MPP++EGB761+L-Arg组高于MPP++EGB761组(P < 0.05)。说明L-Arg可加重MPP+对多巴胺能神经元的损伤作用,EGB761对损伤后的胚胎大鼠中脑原代培养细胞有保护作用,此作用可能是通过抑制诱导型一氧化氮合酶活性降低一氧化氮产生完成的。关键词：银杏叶提取物;一氧化氮;左旋精氨酸;1-甲基-4-苯基吡啶离子; 神经细胞  doi:10.3969/j.issn.1673-8225.2012.20.032     

G-CSF对帕金森病小鼠运动能力和黑质纹状体神经元的影响

目的:探讨粒细胞集落刺激因子(G-CSF)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)所致小鼠帕金森病(PD)模型中脑黑质致密部(SNpc)及纹状体(STR)多巴胺(DA)能神经元的影响。方法:正常健康雄性C57BL/6J小鼠随机分为对照组、MPTP组及MPTP+G-CSF组,采用腹腔注射MPTP法制作小鼠PD模型,MPTP+G-CSF组于最后一次MPTP注射后再腹腔注射G-CSF。爬杆实验观察小鼠的行为学改变;尼氏染色技术观察各组小鼠黑质致密部神经元数量及形态学变化;免疫组织化学法检测酪氨酸羟化酶(TH)在各组小鼠中脑黑质致密部及纹状体的表达; Western Blot法检测各组小鼠中脑TH蛋白的表达量。结果:与对照组相比,MPTP组小鼠较对照组爬杆用时显著延长(P 0. 05),尼氏染色显示黑质致密部神经元数量显著减少(P 0. 05),黑质致密部、纹状体TH表达量显著减少(P 0. 05); MPTP+G-CSF组较MPTP组爬杆用时显著缩短(P 0. 05),神经元丢失减少(P 0. 05),神经元形态较完整;与MPTP组比较,MPTP+G-CSF组黑质致密部、纹状体TH表达水平显著增高(P 0. 05)。结论:G-CSF能够改善PD小鼠运动功能,减少黑质致密部多巴胺能神经元丢失,增加TH表达水平。     

反义寡核苷酸阻断多巴胺转运体表达的大鼠对MPTP反应性的改变

目的 了解反义寡核苷酸阻断大鼠黑质多巴胺神经元多巴胺转运载体表达的效果,观察阻断后大鼠对神经毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)反应性的改变.方法 在立体定向仪下埋管至SD大鼠黑质致密部,4组大鼠分别接受反义寡核苷酸、正义寡核苷酸、错义寡核苷酸及生理盐水注射5d,之后接受MFTP注射,进行行为学、病理免疫组化染色观察多巴胺转运载体表达及多巴胺神经元凋亡.结果 大鼠黑质多巴胺转运载体表达(阳性单位,PU)反义寡核苷酸组(PU=6.65±1.67)较生理盐水对照组(PU=12.41±2.46)、正义寡核苷酸组(PU=11.45±1.17)及错义寡核苷酸组(PU=10.35±2.89)明显降低(免疫组化染色面积减少及强度降低)(P＜0.01);阿扑吗啡诱导的大鼠旋转反义寡核苷酸组较其余3组慢(P＜0.05);其余3组间差异无统计学意义(P＞0.05).反义寡核苷酸组黑质200×视野凋亡细胞个数(21.4±5.6)较其他3组(生理盐水组61.6±19.7,正义寡核苷酸组56.5±16.3,错义寡核苷酸组52.2±12.5)明显减少(P＜0.01).结论 反义寡核苷酸能有效阻断大鼠黑质多巴胺转运载体表达,阻断后大鼠对MPTP毒素反应性减弱.     

Nrf3基因在帕金森病猴模型黑质中表达的研究

本研究目的在于采用 m RNA差异显示技术及改进的硝酸银核酸染色技术 ,寻找 1-甲基 -4 -苯基 -1,2 ,3,6 -四氢吡啶诱导侧与对照侧中脑黑质细胞之间的差异表达基因。用 1-甲基 -4 -苯基 -1,2 ,3,6 -四氢吡啶诱发单侧帕金森病猴模型后 ,将两侧黑质m RNA用 3’端三个锚定引物与 30种随机引物进行逆转录 -聚合酶链反应 ,经聚丙烯酰胺凝胶电泳及硝酸银染色后显示扩增产物条带。结果 :对获得的差异表达基因片段的序列分析、同源性比较及功能分析证明 ,差异基因中的一个 (14 9bp)为 Nrf3基因片段。此结果提示 :Nrf3可能参与小胶质细胞的生长 ,促进对变性神经元的吞噬作用 ,可为从分子水平了解人类帕金森病的发生机理提供依据     

色钉菇乙酸乙酯提取物对帕金森病模型小鼠黑质多巴胺神经元的保护作用

目的:探讨色钉菇乙酸乙酯提取物能否改善帕金森病(PD)小鼠的行为症状以及对黑质多巴胺(DA)能神经元保护作用。方法:以成年C57BL/6小鼠为对象,设置空白对照组、MPTP模型组和100 mg/kg、200 mg/kg、400 mg/kg三个剂量的色钉菇乙酸乙酯提取物的干预组。通过站立次数和爬杆得分等指标评价行为症状,以TH的免疫荧光方法检测存活的黑质DA能神经元,FJC染色法显示黑质致密部变性的DA能神经元。结果:在行为上,实验处理之前各组之间无显著性差异(P0.05);而实验处理后,于站立次数指标上,400 mg/kg剂量干预组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05);在爬杆得分指标上,200 mg/kg组和400 mg/kg组均显著地高于实验对照组(P0.05),但与空白对照组无显著性差异(P0.05)。在TH标记的黑质多巴胺能神经元数量上,只有400 mg/kg组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05)。而FJC标记的变性细胞只在MPTP组、100 mg/kg和200 mg/kg组表达,空白对照组和400 mg/kg组未见FJC阳性细胞。结论:色钉菇乙酸乙酯提取物能显著地改善PD的行为症状和保护黑质DA能神经元,且呈剂量依赖性。     

血管钠肽减轻MPP+所致多巴胺神经元损伤*

 目的：探讨血管钠肽（vasonatrin peptide,VNP）的神经保护效应。方法：原代培养取自小鼠中脑腹侧的多巴胺神经元,暴露于1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium,MPP+）。采用细胞活力分析和免疫荧光染色评价VNP对MPP+神经毒性的影响,并应用多种阻断剂和激动剂探讨VNP神经效应的机制。结果：MPP+造成多巴胺神经元损伤,VNP增强多巴胺神经元的细胞活力,增加神经元轴突数目和长度。VNP还可抑制MPP+造成的神经元β-微管蛋白III解聚。另外,VNP显著升高细胞内cGMP水平。VNP的效应可以被8-Br-cGMP（一种膜通透性的cGMP类似物）所模拟,被HS-142-1［鸟苷酸环化酶偶联的钠尿肽受体（NPR）的阻断剂］或KT-5823［cGMP依赖性蛋白激酶(PKG)的阻断剂］所阻断。结论：VNP通过NPR/cGMP/PKG通路减轻MPP+的神经毒性,提示VNP可能成为一种新的有效药物,治疗帕金森病的神经退行性病变。     

Modeling a sensitization stage and a precipitation stage for Parkinson's disease using prenatal and postnatal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration

Idiopathic Parkinson's disease (PD) is a neurodegenerative disorder of mature and older individuals. Since all aged individuals do not develop PD, predisposing conditions may exist that pair with the stress placed on the basal ganglia during aging to produce the symptoms of PD. In this project we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to test the hypothesis that a sensitization stage and a precipitating stage underlie idiopathic PD. To induce the sensitization stage, pregnant C57BL/6J mice were treated with MPTP (10 mg/kg/day) during gestation days 8–12 to target the emerging fetal nigrostriatal dopamine neurons. For the precipitating stage, the 3-months old offspring were administered MPTP for 7 days, to simulate the changes that occur during aging. The weights and motor activity of the offspring, high performance liquid chromatography (HPLC) striatal dopamine and its metabolites and Western blot for tyrosine hydroxylase (TH) were determined. Offspring exposed to prenatal MPTP showed lower birth weights that eventually recovered. Prenatal MPTP also reduced motor activity by 10–30%, striatal TH by 38%, dopamine by 14%, homovanillic acid by 16.5% and 3-methoxytyramine by 66%. The postnatal MPTP was more potent in the prenatal MPTP-exposed offspring. MPTP at 10, 20 and 30 mg/kg, dose-relatedly, reduced striatal TH by 9.4%, 48.6% and 82.4% in the prenatal-phosphate buffered saline (PBS) mice and by 48%, 78.7% and 92.7% in the prenatal-MPTP groups. More importantly, postnatal MPTP at 10 mg/kg that showed slight effects on DA, DOPAC, HVA and 3-MT in the prenatal-PBS offspring, showed 69.9%, 80.0%, 48.4% and 65.4% reductions in the prenatal-MPTP mice. The study may identify a new model for PD, and the outcome suggests that some cases of idiopathic PD may have a fetal basis in which early subtle nigrostriatal impairments occurred and PD symptoms are precipitated later by deteriorating changes in the nigrostriatum, that would not caused symptoms in individuals with normal nigrostriatal system.     

Effects of Ginkgo Biloba Extract on Free Radical Metabolism of Liver in Mice During Endurance Exercise

This study investigated the effect of Ginkgo biloba extract on Free Radical Metabolism of Liver in mice during endurance exercise. Forty-eight mice were divided into the quiet group and the exercised group. And the two groups were both grouped again, including the control group and the drug-treated group. After exhaustive exercise, the exercised groups were subdivided into the immediate group and the recovery group. The swimming time to exhaustion significantly prolonged in the exercised drug-treated group as compared with the exercised control group (P <0.05); The SOD activity of drug-treated groups significantly increased (P <0.05) as compared with the control groups and MDA content was significantly lower (P <0.05). The SOD activity and MDA content of exercised control groups significantly increased (P <0.05) as compared with the quiet control group. The SOD activity and MDA content of exercised drug-treated groups significantly increased (P <0.05) as compared with the quiet drug-treated group. The results indicated that Ginkgo biloba extract can obviously increase the body''s endurance exercise capacity in mice and delay fatigue; Ginkgo biloba extract can help to increase the activity of the antioxidant enzymes in liver tissue, reduce the lipid peroxidation injury in liver tissue caused by free radicals, improve athletic ability, and promote the recovery process after exercise in mice.     

低剂量MPTP致雌鼠黑质听觉P50诱发电位动态变化的实验研究

目的:功能学角度了解低剂量MPTP对雌鼠黑质听觉P50诱发电位的动态影响及帕金森病动物模型旋转行为的出现与黑质听觉P50诱发电位的关系。方法:45只大鼠随机分为三组:(1)正常对照组;(2)MPTP致帕金森病模型组;(3)生理盐水对照组。动态监测术后第7至14天MPTP型帕金森病动物模型黑质听觉P50诱发电位。结果:MPTP组的P50T/C值在第11~14天均较正常组及生理盐水溶剂对照组(I NT-Saline组)降低,且有统计学意义(P<0.01)。结论:低剂量MPTP可渐进性引起正常雌鼠黑质听觉P50诱发电位降低,且在帕金森病动物模型黑质听觉P50诱发电位降低的同时出现旋转行为。     

Neuroprotective effects of rotigotine in the acute MPTP-lesioned mouse model of Parkinson's disease

Dopamine agonists used to manage Parkinsonian motor symptoms have been suggested to be neuroprotective. The study was designed to assess the neuroprotective potential of the D(3)/D(2)/D(1) dopamine receptor agonist rotigotine in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned (MPTP) mouse model of Parkinson's disease by measuring mesencephalic degenerating neurons using FluoroJade staining and the remaining dopaminergic nerve endings in the striatum using dopamine transporter binding. Continuous administration of rotigotine at a dose of 3mg/kg significantly attenuated MPTP-induced acute cell degeneration in the FluoroJade-staining paradigm. Rotigotine (0.3-3mg/kg) partially protected dopamine nerve endings from MPTP-induced degeneration in a dose-dependent manner. These data suggest that rotigotine, at the doses employed, significantly protected dopamine neurons from degeneration in an acute mouse model of MPTP intoxication.     

虫草素对MPTP诱导的帕金森病小鼠运动与认知能力的影响

目的:探索虫草素(cordycepin)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森病小鼠运动与认知能力的影响。方法:将C57BL/6小鼠每天腹腔注射MPTP (30 mg/kg),连续8 d,以建立帕金森病小鼠模型。HE染色检测虫草素对MPTP诱导小鼠黑质致密部(sub-stantia nigra pars compacta,SNpc)细胞数目的影响;Western blot检测黑质(substantia nigra,SN)内酪氨酸羟化酶(tyrosine hydroxylase,TH)表达量的变化;采用旷场实验(open-filed test,OFT)、自发交替行为(spontaneous alterna-tion behavior,SAB)及水迷宫实验(water maze test,WMT)分别检测虫草素对帕金森病小鼠自发活动、情绪变化和认知行为的影响。结果:HE染色结果表明,虫草素显著抑制由MPTP引起的小鼠SNpc细胞数目减少(P 0.05);Western blot结果表明MPTP显著降低SN内TH的表达量(P 0.05),而虫草素可以逆转这种变化(P 0.05);虫草素可提高小鼠在OFT中的平均速度(P 0.05),并增加小鼠在SAB中的总进臂次数,提高正确率(P 0.05),但对WMT潜伏期并没有明显的影响。结论:虫草素能够减轻MPTP诱导的帕金森病小鼠早期的运动障碍和探索能力降低,但对其记忆障碍并没有明显的影响。     

针刺百会和大椎穴对帕金森病小鼠多巴胺能神经元的保护作用

目的探讨针刺百会和大椎穴对帕金森病(PD)小鼠多巴胺能神经元的保护作用及可能机制。方法C57BL/6雄性小鼠随机分为5组:正常组、模型组、针刺组、美多巴组、针刺结合美多巴组(针美组)。后4组首先腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)建立PD动物模型,后3组造模后采取不同的治疗措施,造模后动物存活14d。采用爬杆法测量小鼠行为学改变,应用高效液相电化学内标法测定前脑单胺类神经递质含量,用免疫组织化学技术检测黑质酪氨酸羟化酶(TH)阳性神经元数目。结果针刺、针美组小鼠爬杆时间较模型组短(P0.05)。与正常组比较,各组动物前脑内多巴胺及其代谢产物3,4-二羟基苯乙酸含量明显降低,3个治疗组与模型组比较虽然无明显差异,但从数值上看降低的程度较模型组低。造模后14d,模型组TH阳性神经元数目较正常组减少(P0.01),3个治疗组也较正常组减少(P0.01),但较模型组增多(P0.05)。结论针刺百会、大椎穴或针刺结合美多巴可改善PD小鼠的行为障碍,这种作用可能与减少多巴胺能神经元的丢失有关。     

银杏叶提取物对糖尿病患者外周血内皮祖细胞GPX及Caspase-3活性的影响

目的:观察银杏叶提取物(GBE)对2型糖尿病(T2DM)患者外周血内皮祖细胞(EPCs)中谷胱甘肽过氧化物酶(GPX)及特异性半胱氨酸蛋白酶-3(Caspase-3)表达的影响。方法:取T2DM患者(实验组)和正常人(对照组)外周血分离单个核细胞,在包被有纤维连接蛋白的培养板中加入含血管内皮生长因子(VEGF)、表皮生长因子(EGF)等的M199培养基,诱导EPCs分化,48h后去除悬浮细胞,继续培养,于培养第6天加入不同浓度GBE(0mg/L、6.75mg/L、12.50mg/L、25.00mg/L、50.00mg/L)干预24h后,用紫外分光光度计检测培养上清液中GPX活性,用酶标仪检测培养细胞Caspase-3活性。结果:相同GBE浓度时,实验组GPX活性较对照组明显降低(P<0.05),Caspase-3活性较对照组明显升高(P<0.05或P<0.01)。结论:GBE对T2DM患者EPCs有保护作用,其机制可能与抗氧化和减少自由基生成有关。