脐血间充质干细胞移植后帕金森病大鼠纹状体内多巴胺含量的变化

背景：目前干细胞移植对帕金森病动物模型的研究多集中在骨髓间充质干细胞和胚胎干细胞方面,关于脐血间充质干细胞移植对帕金森病动物模型的研究相对较少,且未见测量脐血间充质干细胞移植前后多巴胺含量变化的报道。 目的：观察脐血干细胞移植对帕金森病大鼠纹状体内多巴胺含量的影响。 方法：帕金森病模型大鼠随机分成实验组和对照组。将第3代脐血间充质干细胞用Hoechst33258标记后植入实验组大鼠纹状体内,对照组注射磷酸盐缓冲溶液。移植后2,4,8周用免疫荧光双标法检测间充质干细胞的存活、迁移以及胶质纤维酸性蛋白、神经元特异性烯醇化酶、酪氨酸羟化酶和突触素的表达。移植后8周利用高效液相色谱-电化学检测仪检测纹状体多巴胺含量。 结果与结论：脐血间充质干细胞移植后可在大鼠脑内存活,随时间延长迁移范围扩大,分布于纹状体、胼胝体和皮质;胶质纤维酸性蛋白、神经元特异性烯醇化酶、酪氨酸羟化酶都有表达,突触素无表达。多巴胺水平与对照组相比有明显提高 (P < 0.05)。     

大鼠胎脑黑质细胞培养及PD鼠纹状体内悬液微移植研究

目的 探讨如何提高移植存活率和保存供体组织。方法：胎脑黑质细胞原代培养６天后,做免疫组化染色鉴定其生物活性,进行移植质量的人为控制,然后,比较各种移植技术对移植后细胞存活率、胶质细胞反应程度及功能改善的影响。实验动物分Ａ、Ｂ、Ｃ、Ｄ四组。Ａ组为假手术组,Ｂ组为立即单靶点移植组,Ｃ组为立即多靶点微移植组,Ｄ组为培养后多靶点微移植组。结果：Ｂ、Ｃ、Ｄ组术后均有功能改善。Ｃ组各时间点均比Ｂ组改善显著（Ｐ     

STN—DBS对纹状体细胞外液牛磺酸含量的影响

目的探讨丘脑底核脑深部电刺激对猴帕金森病模型纹状体区细胞外液巾牛磺酸含量的影响。方法猴偏侧帕金森病模型,在患侧丘脑底核植入人用脑深部刺激电极,同期猴背部皮下植入脉冲发生器和皮下导线,通过测试后给予有效高频电刺激,利用微透析技术分别在打开脉冲发生器前和开机后的不同时间点在双侧壳核、尾状核头部细胞外液进行取样。对取样标本应用高效液相色谱荧光法检测其中牛磺酸的含量变化。结果电极侧壳核和尾状核的牛磺酸含量在开机后较开机前明显增加（P〈0．05）。结论丘脑底核脑深部刺激后可引起纹状体区细胞外液中牛磺酸的含量明显升高,这可能是丘脑底核脑深部电刺激的作用机制之一。     

帕金森病鼠纹状体内羊膜移植对C-fos表达的影响

目的:观察非多巴胺能组织-羊膜细胞移植是否能纠正帕金森病(PD)鼠纹状体多巴胺(DA)受体的超敏感性。方法:在羊膜细胞植入PD大鼠模型纹状体后第12周,用免疫组化法检测阿朴吗啡诱发的C-fos蛋白。结果:经图像分析,发现成活羊膜细胞移植与死羊膜细胞移植相比,能显著减少移植侧纹状体中C-fos的表达量。结论:成活羊膜细胞移植能够纠正多巴胺受体的超敏现象。而且,多巴胺受体受影响的范围大大超过了细胞移植诱发宿主残存多巴胺神经元再生的范围。     

苯妥英钠在大鼠血液和脑细胞外液药动学研究

目的:分析苯妥英钠在大鼠血浆和脑内药动学变化。方法:采用微透析活体取样和高效液相色谱技术,通过腹腔注射苯妥英钠5 0和10 0mg·kg-1,测定给药后3h内不同时间点大鼠血浆、大脑皮质和海马神经元细胞外液的药物浓度。结果:腹腔注射苯妥英钠10 0mg·kg-1血药浓度显著高于5 0mg·kg-1(P <0 .0 5 ) ,腹腔注射苯妥英钠5 0和10 0mg·kg-1后各时间点海马内药物浓度高于皮质,但统计学比较无显著差异。结论:苯妥英钠迅速进入大鼠脑组织,给药后60～90min达峰浓度。海马内药时曲线下面积略大于皮质,说明给药后海马内药物浓度高于皮质。苯妥英钠半衰期海马长于皮质,说明海马内药物清除速度较慢。腹腔注射苯妥英钠后60min内,血药浓度变化不能反映药物在脑内的代谢趋势,60min后血药浓度降低的速度在一定程度上可以反映脑内药物浓度减低的趋势。     

胎脑黑质脑内移植对PD鼠纹状体内D2RmRNA表达的影响

为了研究胎脑黑质脑内移植后对ＰＤ鼠纹状体神经元Ｄ２Ｒ基因表达的影响，采用核酸斑点杂交技术对太体内Ｄ２ＲｍＲＮＡ进行了连续定量观察，发现胎脑黑质移植物能够使ＰＤ鼠纹状体内过度表达的Ｄ２ＲｍＲＮＡ水平恢复正常。本研究结果提示，Ｄ２ＲｍＲＮＡ过度表达及ＰＤ鼠旋转行为被矫正的程度可能反映出胎脑黑持移植物对宿主形成神经再支配的程度。     

高渗刺激引起培养的大鼠下丘脑星形胶质细胞和C6细胞合成并释放谷氨酸

目的观察高渗刺激对培养的大鼠下丘脑星形胶质细胞或C6细胞合成、释放谷氨酸的影响。方法获取一日龄SD大鼠下丘脑组织，进行星形胶质细胞培养、纯化和鉴定。培养细胞随机分五组：（1）等渗组：用新鲜等渗培养液置换原先的培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（2）高渗组：用320mosMNaCl高渗溶液置换原先的培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（3）甘伯酸（carbenoxolone，CBX，一种缝隙连接阻断剂）＋等渗组和（4）CBX＋高渗组：用含有CBX（终浓度为100mmol／L）的等渗培养液作用1h，后分别用等渗或高渗培养液置换出原培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（5）Ca2＋高渗组：用含有Ca2＋（1000μmol／L）的等渗培养液作用1h，后用高渗培养液置换出原培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。每个亚组5个培养皿。收集各组的细胞，进行抗谷氨酸和抗胶质原纤维酸性蛋白（Glial fibrillary acidic protein，GFAP）的双重免疫荧光染色，Confocal显微镜观察。用反相高效液相色谱荧光测定法测定细胞培养液中谷氨酸的含量。培养的C6细胞分为四组：等渗组，高渗组，CBX＋等渗组和CBX＋高渗组，用于流式细胞仪（flowcytometry，FCM）定量检测细胞内谷氨酸的含量。结果星形胶质细胞内抗GFAP染色的荧光强度在五组问无明显差异。星形胶质细胞内抗谷氨酸染色的荧光强度在等渗组中各时间点无明显变化，高渗组中在刺激1min后表达增加，5min达到高峰，15min恢复到正常。CBX＋高渗组的抗谷氨酸染色荧光强度明显高于CBX＋等渗组和等渗组，一直维持到15min不下降。培养基中的谷氨酸浓度在等渗组各时间点无明显变化，高渗组中谷氨酸浓度从5min到15min明显增加。Ca2＋高渗组和CBX＋高渗组中?     

高渗刺激引起培养的大鼠下丘脑星形胶质细胞和C6细胞合成并释放谷氨酸

目的 观察高渗刺激对培养的大鼠下丘脑星形胶质细胞或C6细胞合成、释放谷氨酸的影响.方法 获取一日龄SD大鼠下丘脑组织,进行星形胶质细胞培养、纯化和鉴定.培养细胞随机分五组:(1)等渗组:用新鲜等渗培养液置换原先的培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(2)高渗组:用320 mosMNaCl高渗溶液置换原先的培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(3)甘伯酸(carbenoxolone,CBX,一种缝隙连接阻断剂) 等渗组和(4)CBX 高渗组:用含有CBX(终浓度为100 mmol/L)的等渗培养液作用1 h,后分别用等渗或高渗培养液置换出原培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(5)Ca2 高渗组:用含有Ca2 (1 000μmol/L)的等渗培养液作用1 h,后用高渗培养液置换出原培养液,将细胞分成5个亚组;分别孵育1、3、5、10和15 min.每个亚组5个培养皿.收集各组的细胞,进行抗谷氨酸和抗胶质原纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)的双重免疫荧光染色,Confocal显微镜观察.用反相高效液相色谱荧光测定法测定细胞培养液中谷氨酸的含量.培养的C6细胞分为四组:等渗组,高渗组,CBX 等渗组和CBX 高渗组,用于流式细胞仪(flow cytometry,FCM)定量检测细胞内谷氨酸的含量.结果 星形胶质细胞内抗GFAP染色的荧光强度在五组间无明显差异.星彤胶质细胞内抗谷氨酸染色的荧光强度在等渗组中各时间点无明显变化,高渗组中在刺激1 min后表达增加,5 min达到高峰,15 min恢复到正常.CBX 高渗组的抗谷氨酸染色荧光强度明显高于CBX 等渗组和等渗组,一直维持到15 min不下降.培养基中的谷氨酸浓度在等渗组各时间点无明显变化,高渗组中谷氨酸浓度从5 min到15 min明显增加.Ca2 高渗组和CBX 高渗组中,培养基中谷氨酸浓度明显低于高渗组,各时间点之间没有明显变化.FCM测定的结果表明高渗或CBX 高渗刺激引起C6细胞内谷氨酸的水平明显上调.结论 高渗刺激可以激发培养的大鼠下丘脑星形胶质细胞合成、释放谷氨酸,CBX不能抑制其合成,但可阻断其释放.     

丘脑底核脑深部电刺激对纹状体区细胞外液谷氨酸含量的影响

目的 探讨长期丘脑底核脑深部电刺激(DBS)对猴偏侧帕金森病模型纹状体区细胞外液中谷氨酸含量的影响.方法 在猴偏侧帕金森病模型的基础上植入DBS系统,刺激电极位于病侧丘脑底核.在给予DBS前后不同时间微透析检测双侧壳核、尾状核细胞外液谷氨酸的含量变化.结果 与开机前比较,DBS后双侧尾状核和壳核的谷氨酸含量均明显升高,1周时升高最显著,1个月以后含量保持稳定;DBS后12个月电极侧壳核谷氨酸含量仍比开机前和非电极侧升高明显(P＜0.05),而尾状核电极侧谷氨酸含量已接近非电极侧(P＞0.05).结论 对猴偏侧帕金森病模型行丘脑底核DBS后,纹状体区细胞外液中谷氨酸的含量明显升高.     

胎脑黑质脑内移植对PD鼠纹状体内D_2RmRNA表达的影响

为了研究胎脑黑质脑内移植后对ＰＤ鼠纹状体神经元Ｄ２Ｒ基因表达的影响，采用核酸斑点杂交技术对纹状体内Ｄ２ＲｍＲＮＡ进行了连续定量观察，发现胎脑黑质移植物能够使ＰＤ鼠纹状体内过度表达的Ｄ２ＲｍＲＮＡ水平恢复正常。本研究结果提示：Ｄ２ＲｍＲＮＡ过度表达及ＰＤ鼠旋转行为被矫正的程度可能反映出胎脑黑质移植物对宿主形成神经再支配的程度。     

脑损伤的干细胞移植治疗研究进展

外伤性脑损伤(traumatic brain injury,TBI)是世界性的多发性疾病,也是死亡率和致残率最高的疾病之一.TBI后生存患者常残留有运动、认知及社会交流障碍,导致患者、家属及社会受到破坏性影响~([1]).     

神经干细胞移植治疗小鼠机械性脑损伤的实验研究

目的探讨神经干细胞移植后的体内存活、增殖与分化,及其对小鼠机械性脑损伤的治疗作用。方法运用牙科钻制作小鼠运动区皮质机械性损伤模型。48只清洁级昆明小鼠,雌雄不拘,体质量为18～20g,按体质量编号随机分为4组:神经干细胞移植组(损伤后原位移植经鉴定确认的原代培养的小鼠神经干细胞)、3T3移植组(损伤后原位移植3T3细胞)、单纯损伤组(损伤后不行神经干细胞移植)和空白对照组(仅施行麻醉),每组12只小鼠。于伤后第3天进行行为学检测;第10、30天行损伤区脑组织nestin及NF200免疫荧光染色,观察神经干细胞生长、分化情况。结果损伤后,获得原代培养的神经干细胞在移植早期贴附于损伤区域且向周边组织呈浸润生长;移植后期Hoechst33342及NF200染色显示损伤区附近可见分化形成的神经元。单纯损伤组小鼠出现偏瘫症状;而神经干细胞移植组小鼠植入神经干细胞后则症状减轻,运动功能明显改善,与其他各组相比差异有显著性意义(P<0.001)。结论神经干细胞移植能够改善小鼠机械性脑损伤后的神经功能状态。     

外源性干细胞移植治疗缺血性脑损伤

全世界脑缺血性卒中患者大约有上千万例,每年都有近百万例新增患者.约半数患者都会遗留不同程度的神经功能缺失.干细胞移植为脑缺血性卒中患者提供了新的治疗方向.神经干细胞移植治疗脑缺血理想目标包括:(1)缺血区受损神经元能被问型神经元替代;(2)诱导局部血管再生使移植神经干细胞长期存活;(3)移植神经元能与周围细胞建立突触联系;f4)胶质细胞辅助移植神经元形成髓鞘.然而以上任何一点的完全实施都是极其困难的.     

Mesenchymal stem cell transplantation changes the gene expression profile of the neonatal ischemic brain

Mesenchymal stem cell (MSC) treatment is an effective strategy to reduce brain damage after neonatal hypoxia–ischemia (HI) in mice. We recently showed that a single injection with MSC at either 3 or 10 days after HI (MSC-3 or MSC-10) increases neurogenesis. In case of two injections (MSC-3 + 10), the second MSC application does not increase neurogenesis, but promotes corticospinal tract remodeling. Here we investigated GFP⁺-MSC engraftment level in the brain using quantitative-PCR analysis. We show for the first time that in the neonatal ischemic brain survival of transplanted MSC is very limited. At 3 days after injection ∼22% of transplanted MSC were still detectable and 18 days after the last administration barely ∼1%. These findings indicate that engraftment of MSC is not likely the underlying mechanism of the efficient regenerative process.Therefore, we tested the hypothesis that the effects of MSC-treatment on regenerative processes are related to specific changes in the gene expression of growth factors and cytokines in the damaged area of the brain using PCR-array analysis. We compared the effect of one (MSC-10) or two (MSC-3 + 10) injections of 10⁵ MSC on gene expression in the brain. Our data show that MSC-10 induced expression of genes regulating proliferation/survival. In response to MSC-3 + 10-treatment a pattern functionally categorized as growth stimulating genes was increased.Collectively, our data indicate that specific regulation of the endogenous growth factor milieu rather than replacement of damaged tissue by exogenous MSC mediates regeneration of the damaged neonatal brain by MSC-treatment.     

细胞移植治疗脊髓损伤的最新进展

一般认为脊髓损伤(spinal cord injury,SCI)造成患者局部甚至全身的瘫痪.大小便失禁和损伤平面以下的感觉消失等功能缺失是永久性的.     

Pergolide potentiates L-DOPA-induced dopamine release in rat striatum after lesioning with 6-hydroxydopamine

Summary. We used intrastriatal microdialysis to study the effect of pergolide, a D₁/D₂ dopamine (DA) receptor agonist on biotransformation of exogenous L-DOPA in hemi-Parkinsonian rats. DA and metabolites were assayed by microbore liquid chromatography. Pergolide (50 μg/kg, i.p) caused a 67% and 87% decrease in striatal EC levels of DA in intact and denervated striatum respectively. In intact striatum but not in denervated striatum, pergolide decreased EC levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) (53% and 42% decrease, respectively). L-DOPA (100 mg/kg, i.p.) produced significant increase in EC levels of DA, DOPAC and HVA in intact and denervated striatum with and without local perfusion of 10⁻⁴ M pergolide. In denervated striatum, L-DOPA-induced DA increase was significantly higher in rats with pergolide. Our results suggest that, in an animal model of Parkinson's disease, pergolide in association with L-DOPA favors the restoration of striatal EC DA levels. Received April 24, 1998; accepted July 23, 1998     

Effect of glutamate receptor antagonists on excitatory postsynaptic potentials in striatum

Glutamate, as the main transmitter of corticostriatal pathway, has a crucial role in the regulation of the activity of striatal cells as well as in pathogenesis of some diseases characterized by striatal malfunction caused by overexcitation of neurons. In the present study, the role of ionotropic excitatory amino acid receptors was investigated in the striatal synaptic transmission. Using conventional intracellular electrophysiological methods in brain slices, we have investigated the effects of the N-methyl-D-aspartate (NMDA) antagonist (±) 2-amino-5-phosphono-valerate (APV) and the α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist (±) 1-(4-aminophenyl)-3-methyl-carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655) on the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of corpus callosum. The AMPA antagonist significantly decreased electrically evoked responses and a weak inhibition was also observed after APV application. The results were compared to similar data obtained in a cortical slice study.     

神经干细胞发育调控及移植疗效影响因素分析

神经干细胞由于其多分化的潜能被广泛运用于临床治疗各种疾病,其治疗方式主要有两种:一种是植入外源性神经干细胞[1];另一种是促进内源性神经干细胞增殖[2].无论哪种方式,弄清控制神经干细胞增殖、分化、迁移以及影响其疗效的相关因素都极为重要.     

Effects of gliosis on dopamine metabolism in rat striatum

Neuroimplantation is inevitably accompanied by gliosis. Although graft-induced trophic effects on host neurons may be mediated by glial cells, the effects of gliosis on dopamine (DA) metabolism remains unclear. To examine these effects, basic fibroblast growth factor (bFGF) was directly infused into the striatum of 12 male rats (250–280 g). One week later, substantial gliosis was demonstrated in the infused striatum by immunochemical staining for glial fibrillary acidic protein (GFAP) and quantified by GFAP Western blot analysis. One week after bFGF infusion, extracellular DA and its metabolites were measured by in vivo microdialysis using HPLC. Infusion of -DOPA through the dialysis probe resulted in a 60% reduction in the -DOPA-induced DA peak in the gliotic striatum compared with the normal side. After -DOPA infusion, dihydroxyphenylacetic acid (DOPAC) levels were similar between the gliotic and normal striatum. In contrast, homovanillic acid (HVA) levels were 26% higher in the gliotic striatum. Enzyme assays demonstrated that aromatic -amino acid decarboxylase activity was unchanged in the gliotic striatum, but both MAO-A and MAO-B activities increased by 23% and 21%, respectively. These results suggest that the reduced striatal DA peak in the gliotic striatum after -DOPA administration was due to accelerated DA catabolism through enhanced MAO activity. The bFGF-induced striatal gliosis may serve as a model to study neurotransmitter metabolism in the gliotic brain caused by disease processes, aging, or tissue grafting.     

Effects of NADH on dopamine release in rat striatum

Nicotinamide adenine dinucleotide (NADH) may be utilized for the synthesis and regeneration of tetrahydrobiopterin (BH(4)), which in turn is an essential cofactor for tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine (DA). NADH has been reported to relieve some of the symptoms of Parkinson's disease, presumably by altering dopaminergic function. The present study examines the efficacy of NADH in influencing DA activity in the rat striatum. In striatal slices, NADH (350 microM) significantly increased basal DA and DOPAC efflux and caused a 2-fold increase in the DA overflow evoked by high KCl (25 mM). Tissue levels of BH(4), basal BH(4) efflux, and KCl-evoked BH(4) overflow were unaffected by NADH, as was [(3)H]DA uptake into striatal synaptosomes. In contrast to the effects of NADH on DA function in vitro, no effects were observed when NADH was administered systemically. NADH (10 or 100 mg/kg, s.c.) did not influence the tissue content of DA, 5-HT, or their metabolites in the midbrain or striatum, nor did it alter DA extracellular concentrations. These results indicate that NADH can increase DA release from striatal slices, although we are as yet unable to detect this effect in vivo.