GDNF转基因成纤维细胞脑内移植治疗帕金森病大鼠模型

为了观察表达胶质细胞系源性神经营养因子 (GDNF)的基因工程细胞在脑内移植后对帕金森病大鼠的治疗作用 ,将 6-羟基多巴胺脑内定位单侧 (损伤侧 )注射制备大鼠帕金森病模型。随机将动物分为实验组 (2 0只 )和对照组 (15只 ) ,分别植入GDNF和 Lac Z转基因的原代培养大鼠成纤维细胞于损伤侧纹状体内 ,动态观察动物单侧旋转行为以及多巴胺及其代谢产物 3 ,4-二羟苯乙酸和高香草酸含量的变化。结果表明 :实验组帕金森病大鼠单侧旋转行为明显减少 ,为移植前旋转圈数的 (64 .8± 5 .8) % (P<0 .0 5 ) ;其纹状体内多巴胺含量显著提高 ,恢复至移植前水平的 (14 .5± 2 .2 ) % (P<0 .0 5 ) ,而对照组仅为 (1.92± 0 .15 ) % ;转基因细胞在脑内可存活 1年以上。本研究结果提示 GDNF基因治疗对大鼠帕金森病具有潜在的临床应用价值。     

止颤汤干预神经干细胞移植帕金森病大鼠脑内多巴胺及其代谢产物的变化

背景：如何促进脑内多巴胺含量的增加以及减少多巴胺的代谢,是治疗帕金森病的热点所在。 目的：从多巴胺代谢途径角度观察止颤汤对神经干细胞移植帕金森病大鼠的脑黑质中多巴胺及其代谢产物含量的变化。 方法：以大鼠脑立体定位和1-甲基-4-苯基-1,2,3,6-四氢吡啶建立帕金森病大鼠模型。应用高效液相色谱法测定帕金森病大鼠中脑多巴胺及其代谢产物的含量。 结果与结论：止颤汤可以提高神经干细胞移植后帕金森病大鼠中脑多巴胺及其代谢产物双羟苯乙酸的含量,但对代谢产物高香草酸无明显影响。通过促进帕金森病大鼠干细胞移植后神经干细胞的存活,使之定向分化为多巴胺能神经元并分泌多巴胺,同时抑制多巴胺分解达到治疗作用。     

益精养血方对帕金森病大鼠多巴胺能神经元Caspase-3表达的影响

目的研究益精养血方对帕金森病(Parkinson's disease,PD)大鼠多巴胺能神经元Caspase-3表达的影响。方法将微量6-羟多巴胺(6-OHDA)立体定向注射于SD大鼠中脑右侧黑质以建立大鼠帕金森病模型,将造模成功的实验动物随机分为模型组、实验(中药)组,每组6只;另纳入6只正常大鼠为正常组。正常组和模型组大鼠生理盐水灌胃,实验组大鼠益精养血方灌胃,各30天。灌胃结束两周后进行行为学检测,免疫组织化学方法观测黑质酪氨酸羟化酶(TH)、Caspase-3的表达。结果实验组大鼠的旋转圈数比治疗前明显减少(P<0.05),实验组损毁侧与健侧黑质TH阳性细胞数量百分比较模型组显著提高(P<0.05),实验组损毁侧黑质Caspase-3表达的OD值比模型组显著降低(P<0.05)。结论益精养血方可使帕金森病大鼠多巴胺能神经元Caspase-3的表达降低,明显抑制神经细胞凋亡。     

6-羟基多巴胺单侧两点注射法构建的帕金森病模型鼠

背景：帕金森病动物模型的建立对帕金森病的临床、基础实验研究中有重要作用,同时其稳定性也直接影响到研究的结果。 目的：评价6-羟基多巴胺单侧两点注射法构建的帕金森病模型大鼠行为及病理变化。 方法：SD大鼠62只随机分为实验组50只,正常组12只,采用脑内立体定向,将6-羟基多巴胺注入实验组大鼠右侧黑质致密部和中脑腹侧被盖区以建立帕金森病模型,观察大鼠行为变化、酪氨酸羟化酶及脑内多巴胺含量改变。 结果与结论：2周后实验组经阿朴吗啡诱导后,有22只向左侧旋转速度>7 r/min,2周与4周之间大鼠的旋转圈数差异无显著性意义(P > 0.05)。模型组中酪氨酸羟化酶及脑内多巴胺含量明显减少。说明应用6-羟基多巴胺单侧两点注射可成功建立行为及病理相似的帕金森病大鼠模型。     

胃腔内促性腺激素释放激素类似物对大鼠胃和十二指肠生长抑素细胞功能的影响

目的　研究胃腔内促性腺激素释放激素类似物 (GnRH A)对大鼠胃和十二指肠生长抑素免疫阳性细胞及血液、胃液中生长抑素含量的影响。　方法　应用免疫组织化学ABC和ELISA方法分别检测大鼠胃和十二指肠生长抑素阳性细胞的密度及血液、胃液中生长抑素的含量。　结果　胃腔注射GnRH类似物后 ,胃壁内单位面积生长抑素阳性细胞数为 2 6 6± 3 893,与对照组 4 8 3± 6 0 19相比具有差异 (P <0 0 1) ,十二指肠单位面积生长抑素阳性细胞数为 5 1 7± 2 2 14 ,与对照组 5 8 5± 4 4 5 4相比具有差异 (P <0 0 1)。血液中生长抑素ELISA检测的A值为 0 15± 0 0 18,与对照组 0 2 7± 0 0 36相比具有差异 (P <0 0 1) ,胃液中A值为 0 32± 0 0 4 9,与对照组 1 0 2 2±0 36 9相比具有差异 (P <0 0 1)。　结论　外分泌的GnRH对大鼠胃和十二指肠中生长抑素的分泌起显著的抑制作用。     

脑室内注射BDNF抗体对大鼠海马NOS表达的影响

探讨脑室内注射脑源性神经营养因子 (BDNF)抗体阻断内源性BDNF对大鼠海马一氧化氮合酶 (NOS)阳性神经元的影响。脑室内注射BDNF抗体一周后 ,采用Morris水迷宫进行行为检测 ;并用NADPH 黄递酶组化染色方法观察海马NOS阳性神经元数目的变化。与对照组相比 ,实验组大鼠空间学习和记忆能力明显下降 (P <0 0 1) ;实验组大鼠海马CA1区NOS阳性神经元数目 (38 37± 5 2 3)明显少于对照组 (4 9 5 3± 5 74 ) (P <0 0 1) ;实验组DG区NOS阳性神经元数目 (4 8 77± 5 5 1)明显少于对照组 (6 0 4 0± 7 39) (P <0 0 1)。脑室内注射BDNF抗体可导致大鼠空间学习记忆能力下降 ,海马NOS阳性神经元数目减少 ,提示BDNF对学习和记忆的影响可能与海马NOS阳性神经元数目的变化有关     

大鼠肾上腺髓质脑内移植后中枢多巴胺系统的代谢与功能研究

向Wistar大鼠脑内微量注射6羟多巴胺(6-OHDA)破坏一侧黑质,造成Apomorphine诱发的旋转模型。将同系大鼠肾上腺髓质植入损伤侧尾状核,两个月后观察行为改变。其中4/10停止旋转,6/10旋转次数明显减少。用高效液相电化学检测器(HPLC-ED)测损伤侧端脑多巴胺(DA)含量,发现比健侧减少37％(P<0.01),其代谢产物高香草酸(HVA)也明显减少(P<0.05)。经肾上腺髓质移植后,DA含量仅增长9％,代谢产物无变化,去甲肾上腺素(NE)含量明显增高(P<0.05)。此外还发现损伤侧脑突触体对DA的摄取明显增加,DA_2受体结合活性显著增高(P<0.01),这些变化在脑移植大鼠组未能测到。     

HBV核心与前S1融合蛋白疫苗对HBV转基因鼠的免疫治疗效果观察

目的　观察乙型肝炎 (乙肝 )病毒核心与前S1融合蛋白疫苗 (HBVCS1)在HBV转基因小鼠中的免疫治疗效果。方法　 6只HBV转基因小鼠分成 2组 ,在 0周和 3周时实验组小鼠腹腔内注射HBVCS1与免疫佐剂 (弗氏完全佐剂 弗氏不完全佐剂 ) ,对照组仅注射弗氏完全佐剂与磷酸盐缓冲液PBS。采用 3H TdR掺入法检测转基因鼠脾淋巴细胞特异性增殖反应 ,以酶联免疫吸附试验检测血清中的乙肝病毒表面抗原HBsAg,采用荧光定量PCR检测血清中的HBVDNA。结果　实验组 (1 99± 0 35 )转基因鼠脾细胞抗原特异性增殖反应明显高于对照组 (1 5 6± 0 15 ) ;实验组第 6周 (3 5 8±0 80 )与第 9周 (3 4 5± 0 70 )血清中HBsAg终点滴度与第 0周 (4 36± 1 0 4 )及第 3周 (4 81± 0 98)以及同一时间实验组与对照组之间HBsAg终点滴度均差异有显著意义 ;实验组小鼠第 6周 (4 0 2± 0 2 9)和第 9周 (3 5 8± 0 17)血清中HBVDNA含量明显低于免疫前 (5 5 5± 0 88) ,而对照组各批血清无此差别。结论　HBVCS1蛋白疫苗在HBV转基因小鼠中能诱导特异性免疫 ,并具有抑制HBVDNA复制和HBsAg表达的治疗效果     

益精养血方对帕金森病大鼠多巴胺能神经元凋亡的影响

目的:研究益精养血方对帕金森病(Parkinson disease,PD)大鼠多巴胺能神经元凋亡的影响。方法:采用6-羟多巴胺制备的大鼠PD模型,实验分为模型组、实验(中药)组和正常组。正常组和模型组大鼠生理盐水灌胃,实验组大鼠益精养血方灌胃,各30 d。灌胃结束2周后行行为学检测,免疫组织化学方法观测黑质酪氨酸羟化酶(TH)、Bcl- 2、Caspase-3的表达,免疫电镜技术观察黑质多巴胺能神经元结构变化。结果:实验组大鼠的旋转圈数比治疗前明显减少;实验组损毁侧与健侧黑质TH阳性细胞数量较模型组显著提高;实验组损毁侧黑质Caspase-3表达的D值比模型组显著降低,而Bcl-2的表达与Caspase-3相反;免疫电镜观察TH免疫反应产物表达于黑质DA能神经元高尔基复合体质膜面及胞质内,电子密度较高,在正常组中多见,实验组较少,而模型组很少见。结论:益精养血方对PD大鼠多巴胺能神经元凋亡有明显抑制作用。     

不同电针刺激强度和频率对大鼠脑内单胺类神经介质有不同的影响

比较了两种不同电针刺激强度(负载电压峰-峰值3伏,6伏)和频率(10赫兹,200赫兹)对大鼠不同脑区内5-羟色胺(5-HT)及其代谢产物5-羟吲哚乙酸(5-HIAA)、去甲肾上腺素(NA)和多巴胺(DA)水平的影响。70只体重为250克左右的健康雄性大鼠随机分为五个实验组:Ⅰ对照组,Ⅱ低     

骨髓基质细胞对离体Parkinson鼠脑片单胺类神经递质的影响

为探讨骨髓基质细胞对离体Parkinson鼠脑片单胺类神经递质的影响,本研究通过建立新生大鼠离体“Parkinson鼠病”模型,取成年大鼠骨髓,培养、分离、纯化骨髓基质细胞,将骨髓基质细胞(MSCs)与离体脑片联合培养,应用高效液相法观察了脑片和培养液中多巴胺及其代谢产物3,4-二羟苯乙酸(DOPAC)和高香草酸(HVA)含量的变化。结果显示,经MPTP代谢产物mpp+损伤的脑片多巴胺含量与正常组、联合培养组相比明显减少(P<0.01),而mpp+组培养液内DOPAC和HVA含量也减少(P<0.05)。但联合培养组与正常组相比DA,DOPAC和HVA的变化无显著差异。以上结果提示,联合培养的骨髓基质细胞对mpp+毒性损伤的多巴胺能神经元具有保护作用,该模型也为在脑片上测量单胺类神经递质提供了可靠的方法。     

Dopamine and preparatory behavior: II. A neurochemical analysis

Changes in the activity of dopamine-containing systems in relation to preparatory and consummatory feeding responses were investigated. In Experiment 1 rats were conditioned to associate food delivery with the presentation of a conditional stimulus (CS+). When sacrificed after exposure to the CS+ alone on a test trial, the ratio of the dopamine metabolite 3,4-dihydroxyphenylacetic acid to dopamine (DOPAC/DA ratio) was increased significantly in the nucleus accumbens. A similar trend in the ratio of homovanillic acid to dopamine (HVA/DA ratio) was also observed. Similar increases were observed in the striatum, but these were not statistically significant. In contrast, no increases were observed in the DOPAC/DA ratio or the HVA/DA ratio in either brain region when rats were permitted to consume an unsignaled meal for 7 min. These findings suggest that activation of dopamine terminals in the nucleus accumbens occurs during the anticipation of a meal, at which times the rat is engaged in preparatory feeding behaviors, but does not accompany the performance of short bouts of consummatory feeding behavior.     

Feeding and hypothalamic stimulation increase dopamine turnover in the accumbens

The hypothesis that the dopaminergic system plays a role in feeding behavior was tested in three experiments. First, microdialysis was performed in the nucleus accumbens (NAC) at 20 min intervals during free feeding in rats at 80% of normal body weight. Extracellular concentration of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) increased significantly during eating indicating an increase in DA turnover. Second, microdialysis samples were collected from the NAC during bar pressing with a) a signal light on and food available, b) the light on but no food available, c) neither light nor food. Only when food was available did extracellular DA, DOPAC and HVA increase significantly. This increase in DA turnover occurred in the accumbens but not in the ventral striatum. Third, electrical stimulation of the perifornical lateral hypothalamus (LH) that was capable of inducing feeding increased extracellular DA, DOPAC and HVA in the NAC. This occurred whether the animal had food to eat or not. The effect of LH stimulation on DA turnover resembled the effects of free feeding and operant feeding in Experiments 1 and 2. Perifornical LH stimulation did not increase dopamine turnover in the ventral striatum. The results show that perifornical LH stimulation activates the mesolimbic dopamine system and that dopamine release in the accumbens is involved in feeding. The increase in dopamine turnover outlasted the consummatory act. This suggests that accumbens dopamine may be related to sensory input, feeding reflexes, food reward or memory processes and not just to the consummatory act itself.     

The long-term effects of neurotoxic doses of methamphetamine on the extracellular concentration of dopamine measured with microdialysis in striatum.

The extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the striatum were measured by in vivo microdialysis in freely moving rats one week after the animals were treated with neurotoxic doses of methamphetamine. Methamphetamine produced a marked depletion of striatal DA measured in postmortem tissue, and in the extracellular concentrations of DOPAC, HVA and 5-HIAA. In contrast, the resting extracellular concentration of DA in striatum was the same as in saline-pretreated controls. Furthermore, methamphetamine-pretreated rats were able to increase their concentration of extracellular DA to the same extent as controls in response to a (+)-amphetamine challenge. It is suggested that this adaptive response is probably responsible, at least in part, for the absence of obvious behavioral deficits in animals exposed to neurotoxic doses of methamphetamine.     

Comparison of the effects of intracerebrally administered MPP+ (1-methyl-4-phenylpyridinium) in three species: microdialysis of dopamine and metabolites in mouse, rat and monkey striatum

Intracerebral microdialysis in 3 awake species allowed the measurement of the basal output of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole-acetic acid (5-HIAA) from rat and mouse striatum and monkey caudate in vivo. The DOPAC/HVA ratios in dialysates from mouse and rat striatum were about 1 and 2 respectively, but only 0.09 in monkey caudate dialysates. The extracellular levels of the metabolites correlated well with reported tissue levels, while extracellular DA levels were 3 orders of magnitude lower than tissue concentrations. The effects of the intracerebrally administered dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) were essentially similar in the 3 species. In all cases an immediate, massive release of DA was accompanied by a pronounced decrease in the output of the metabolites. Basal DA release was no longer detectable 5-12 h after MPP+ administration and a second MPP+ perfusion failed to increase the release of DA.     

In vivo release of dopamine and its metabolites following a direct infusion of L-dopa into the caudate nucleus of awake, freely behaving rats using a push-pull cannula

In vivo release of dopamine (DA) and its metabolites were determined following a direct infusion of 3,4-dihydroxyphenylalanine (L-DOPA) through a push-pull cannula in the caudate nucleus of unanesthetized, freely behaving rats. L-DOPA infusions increased the release of DA and dihydroxyphenylacetic acid (DOPAC) beginning with 10(-5) M L-DOPA, while homovanillic acid (HVA) was released consistently only following 10(-3) M L-DOPA. Maximal release of DA preceded that of DOPAC which preceded that of HVA. No salient changes in 5-hydroxyindoleacetic acid or behavior were observed following any L-DOPA dose.     

鼠胚神经干细胞移植对帕金森模型大鼠的治疗作用

背景：干细胞移植是治疗帕金森的有潜力的方法之一。 目的：观察神经干细胞纹状体移植对帕金森模型大鼠旋转行为及脑内多巴胺含量的影响。 方法：采用6-羟基多巴胺定点注射毁损黑质纹状体的方法构建帕金森大鼠模型;向造模成功的大鼠纹状体内分别移植1×106(共计20 μL)的第3代胚鼠神经干细胞或等量生理盐水。 结果与结论：神经干细胞移植后,帕金森大鼠的旋转行为明显改善。干细胞移植后3周,免疫组化检测发现移植干细胞的帕金森大鼠脑黑质部位酪氨酸羟化酶阳性细胞数增多,纹状体内可见酪氨酸羟化酶阳性细胞;荧光显微镜下观察发现Hoechst 33324d标记神经干细胞在移植针道附近最为密集,并向远隔部位迁徙。干细胞移植后8周,高效液相色谱检测显示移植干细胞的帕金森大鼠纹状体内多巴胺含量明显增高(P < 0.01)。说明神经干细胞脑内移植能够减轻6-羟基多巴胺引起的大鼠中脑黑质多巴胺能神经元的损伤,改善大鼠的旋转行为。     

神经干细胞联合胶质细胞源性神经营养因子脑内移植对Parkinson病大鼠纹状体内多巴胺含量的影响

将原代培养的胚鼠(E14.5)腹侧中脑神经干细胞(NSCs)单独或联合胶质细胞源性神经营养因子(GDNF)移植入Parkinson病(PD)大鼠纹状体内,术后各组动物存活一段时间,观察旋转行为后处死,取纹状体组织做高效液相色谱检测多巴胺(DA)含量。探讨NSCs单独或联合GDNF脑内移植对PD大鼠纹状体内DA含量的影响。结果显示:与PD模型相比,NSCs单独或联合GDNF脑内移植均能提高PD大鼠纹状体内的DA含量(P<0.05);在术后30d和60d,DA含量在GDNF+NSCs组的增加幅度比NSCs组更加明显,动物旋转行为亦得到明显改善(P<0.05)。上述结果表明,NSCs单独或联合GDNF移植均对PD大鼠有一定的治疗作用,而NSCs联合GDNF移植的治疗效果更好。