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。     

别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

目的 探讨别孕烯醇酮（APα）对帕金森病（PD）小鼠黑质-纹状体多巴胺能系统及行为学的影响及可能的分子机制。方法 90只3月龄体重为20~25 g雄性C57BL/6小鼠1侧纹状体被注射6-羟多巴胺（6-OHDA）复制小鼠PD模型,再给予APα及γ-氨基丁酸A受体（GABAAR）拮抗剂荷包牡丹碱（Bic）。采用ELISA检测血清及大脑皮质APα含量和纹状体多巴胺水平,免疫组织化学法检测黑质多巴胺能神经元及其纹状体纤维投射数量的变化,Western blotting检测中脑胞膜GABAAR、胞质及胞核各蛋白水平的变化,并通过免疫共沉淀验证它们之间的相互作用,观察小鼠行为学的变化。结果 PD小鼠大脑皮质内源性APα水平显著降低,黑质多巴胺能阳性神经元及其纹状体纤维投射含量下降,胞膜、胞质和胞核各蛋白的水平也明显下降,小鼠的运动功能发生障碍。在给予外源性APα处理后,上述情况均有所改善。但在应用Bic后,上述调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号呈现相反的变化,免疫共沉淀结果显示,蛋白之间存在相互作用。结论 外源性的APα通过增加其内源性水平调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号通路促进PD模型小鼠黑质-纹状体多巴胺能神经系统和小鼠行为学的改善。     

丙戊酸钠对视网膜色素变性小鼠光感受器细胞的保护作用

观察在视网膜变性小鼠（retinal degeneration mouse,rd小鼠）视网膜色素变性过程中,丙戊酸钠（valproate sodium,VPA）是否对光感受细胞具有保护作用。取48只新生rd小鼠,随机分为VPA组和正常对照组,每组24只。（1）VPA组,出生后每日以VPA100mg/kg的剂量进行腹腔注射;（2）对照组,出生后每日腹腔注射与VPA相同体积的生理盐水。于出生后7天、14天、21天、28天分别将VPA组和对照组的6只小鼠处死,取眼球做冰冻切片,行HE染色,对两组视网膜光感受器细胞数进行计数,并测量外核层厚度;用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL法）检测凋亡细胞数,并对凋亡细胞数目进行统计分析。结果显示：（1）出生后7天VPA组光感受器细胞数和外核层厚度与对照组相比并无统计学差异,出生后14天、21天、28天,VPA组光感受器细胞和外核层厚度多于对照组,两者具有统计学差异（P〈0.05）;（2）两组光感受器细胞均于出生后7天开始出现凋亡,在出生后7天、14天VPA组外核层凋亡数少于对照组,两者具有统计学差异。提示VPA具有对抗视网膜色素变性小鼠光感受器细胞凋亡的作用,能延缓光感受器细胞的进行性退变。     

GDNF在坐骨神经损伤后大鼠L4—6背根节神经元的表达及变化

目的 观察坐骨神经夹伤后不同时间点,胶质细胞源性神经营养因子（ＧＤＮＦ）在成年大鼠Ｌ４～６背根节（ＤＲＧ）中的分布,探讨ＧＤＮＦ对感觉神经元损伤的反应。方法 成年大鼠右侧坐骨神经夹伤后,分别取伤后１、５、７、１０、１４、２８和５６ｄＬ４～６背根节,行抗ＧＤＮＦ多克隆抗体免疫组织化学ＡＢＣ法染色,之后对染色结果进行图像分析。结果 ＧＤＮＦ免疫反应存在于坐骨神经夹伤后１、５、７、１０、１４、２８和５６ｄ成年大鼠Ｌ４～６背根节神经元中,图像分析结果表明,伤后１、３、５和７ｄ的损伤侧背根节神经元的平均光密度大于对照侧背根节神经元。结论 大鼠坐骨神经夹伤后,ＧＤＮＦ在背根节Ｌ４～６感觉神经元中有一定量的变化,且伤后１周内ＧＤＮＦ在伤侧背根节神经元中的表达增强。     

丙戊酸钠对癫痫患儿糖代谢影响的临床研究

目的：观察癫痫患儿服用VPA后体重、体重指数（BMI）、血糖、胰岛素、胰岛素抵抗的变化,了解VPA对糖代谢的影响。方法：选择正常儿童65例为对照组,设置未服VPA治疗的癫痫患儿65例为Ⅰ组,65例癫痫患儿经治疗VPA3个月后为Ⅱ组,已单服VPA治疗达6个月者65例为Ⅲ组。分别检测体重、体重指数（BMI）、血糖、胰岛素、胰岛素抵抗指数。结果：治疗3个月的患者（Ⅱ组）,体重、体重指数及空腹胰岛素水平较治疗前明显增高（P〈0.01）;空腹血糖水平则明显降低（P〈0.05）。治疗6个月的患者（Ⅲ组）,体重、体重指数更为增高,但空腹胰岛素水平较治疗3个月者下降（P〈0.05）;空腹血糖水平较治疗3个月者稍有增高,但无统计学意义。胰岛素抵抗指数三组间变化不大。治疗6个月的患者,食欲增加者有47例,而治疗前食欲增加者仅20例,其差异有统计学意义（P〈0.05）。结论：服用VPA可导致癫痫患儿食欲增加、除不存在胰岛素抵抗外,体重、血糖和高胰岛素水平在治疗3月后已可产生明显变化。     

丙戊酸钠在精神科临床中的应用

目的：了解丙戊酸钠在临床上的治疗效果。方法选取我院在2013年1月~2014年1月这1年时间内收治的精神科患者共160例,随机分为对照组及观察组,每组80例。对照组采用常规模式治疗,观察组采用丙戊酸钠药物治疗。统计患者使用药物后的不良反应情况,了解这类药物临床应用于不同精神疾病的作用。结果丙戊酸钠能够有效缓解患者不同精神疾病症状,减轻躁动、抑郁等不良情绪的程度,但临床使用中易出现头晕头痛、血小板减少、胃肠道不适等不良反应。结论丙戊酸钠对于精神科患者而言临床效果较好,能够减轻患者精神疾病程度,但使用时应注意对患者不良反应的预防与治疗,提升临床用药有效性。     

移植BDNF和GDNF基因修饰的hMSCs对大鼠大脑中动脉阻塞的影响

 目的：构建脑源性神经营养因子（BDNF）和胶质细胞源性神经营养因子（GDNF）基因的非病毒表达载体,用脂质体法转染人骨髓间充质干细胞（hMSCs）,观察其对大鼠大脑中动脉阻塞（MCAO）模型的影响,探索移植转基因修饰的hMSCs治疗脑血管疾病的可行性。方法：构建高效非病毒表达载体,用脂质体法转染获得高表达2种神经营养因子的hMSCs。建立大鼠MCAO模型,建模后24 h经股静脉进行转基因hMSCs移植,并以磷酸盐缓冲液(PBS)和hMSCs为对照。用脑梗死体积计算、体重变化、行为学评测等指标对大鼠脑损伤程度进行评估,通过大鼠脑组织观察和病理切片对脑组织损伤以及细胞的迁移分化情况进行分析。结果：经股静脉转基因hMSCs移植能够提高大鼠MCAO后的感觉运动功能,减小脑梗死体积,与PBS对照组相比有显著差异;与hMSCs治疗组相比,治疗效果较好且稳定。移植的细胞在脑损伤区域有少数存活但未见分化现象。结论：经静脉移植脂质体介导、GDNF和BDNF基因修饰的hMSCs,可促进缺血脑组织的损伤修复,效果较好,为非病毒载体在干细胞相关转基因治疗的应用提供了理论依据。本研究表明,MSCs的作用不依赖干细胞的分化和神经元的替换,而可能与其分泌细胞因子对抗脑损伤并促进神经修复有关,在MSCs中转入特定的外源性神经营养因子可加强这一作用。     

GDNF的基因结构、信息传递及神经保护作用

胶质细胞系源性神经营养因子 (GDNF)是 TGF-β家族的一个新亚族 ,该亚族还有新发现的三个成员 :Neurturin (NTN)、 Persephin (PSP)和 Artem in (ART) ,它们均不程度地对多巴胺神经元、脊髓前角运动神经元、背根神经节、颈上神经节以及肾等非神经元细胞都具有生长促进作用。 GDNF基因位于 5 p12 - 13. 1,全长约 2 8～ 30 kb,由三个外显子和两个内含子构成 ,转录后形成 4.6 kb的 m RNA,其中编码区全长 6 36 bp,超始密码子在第二个外显子上 ,编码的 GDNF前体蛋白为 2 11个氨基酸残基 (其中信号肽 19个氨基酸 ) ,酶解后形成 134个氨基酸的成熟多肽。 GDNF受体系统由膜外糖磷酯酰肌醇 (GPI)偶联的直接与 GDNF结合的受体 GFR (GDNFfam ily receptor)和跨膜的酪氨酸激酶 Ret两部分构成。研究表明将 GDNF向纹状体、黑质或脑室内直接注射 ;胚胎中脑组织用 GDNF处理后植入脑内 ;GDNF转基因工程细胞脑内移植 ;用腺病毒、腺相关病毒介导的 GDNF c D-NA直接注入脑内等不同方式给予帕金森病模型动物 ,可以改善这些动物的 PD症状     

丙戊酸钠对人脑胶质瘤细胞系SHG-44增殖分化的影响

目的 研究丙戊酸钠对培养人脑胶质瘤细胞系SHG-44细胞的增殖抑制和分化诱导作用。方法 以0.25、0.5、1.0、2.0、4.0mmol/L丙戊酸钠处理SHG-44细胞,用MTT比色、流式细胞术、光镜观察、免疫组化染色等方法检测处理细胞。结果 SHG-44细胞经丙戊酸钠处理后：（1）细胞增殖受到明显抑制,并具有时间,剂量依赖性;（2）细胞周期受阻,S期细胞比例减少,G1期细胞比例增多;（3）胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）表达增强。结论 丙戊酸钠能抑制人脑胶质瘤细胞系SHG-44细胞的增殖,1.0mmol/L丙戊酸钠能诱导SHG-44细胞发生明显分化。     

GDNF和HSV-GDNF对培养的大鼠脊髓运动神经元缺氧后的Bcl-2表达的影响

目的:观察细胞胶质源性神经营养因子(Glial cell line-derived neurotrophic factor,GDNF)和单纯疱疹病毒介导的GDNF(GDNF transformed by herpes simplex virus vector,HSV-GDNF)对体外培养的大鼠脊髓运动神经元缺氧-复氧时的作用和Bcl-2表达的影响。方法:观察缺氧2h、4h和缺氧4h后恢复供氧24、72h时,脊髓运动神经元存活数,并用抗Bcl-2抗血清行免疫组织化学染色,对Bcl-2免疫反应阳性神经元作平均光密度分析。结果:经GDNf、HSV-GDNF孵育的脊髓运动神经元缺氧-复氧后Bcl-2表达较对照组明显增强,神经元损伤程度减轻,神经元存活数明显高于对照组,且HSV-GDNF组的效果更好。结论:GDNF、HSV-GDNF对缺氧-复氧的大鼠脊髓运动神经元有保护作用,HSV-GDNF比GDNF更能增强缺氧-复氧后脊髓运动神经元Bcl-2的表达,提高神经元存活数,抑制缺氧后神经元的死亡。     

BDNF在Parkinson病小鼠的表达及丙戊酸盐对其影响的研究

为观察Parkinson病(PD)小鼠脑源性神经营养因子(BDNF)含量的变化及丙戊酸盐(valproate,VPA)对BDNF表达的影响,探讨VPA对PD病神经元的保护作用,本研究采用C57BL/6小鼠1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methy1-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)法建立PD模型。小鼠随机分为4组:盐水对照组(NS+NS)、模型组(NS+MPTP)、模型给药组(VPA+MPTP)和单独给药组(VPA+NS)。MPTP造模方法为每日颈部皮下注射MPTP(20 mg/kg),连续8 d。VPA在MPTP造模前1 d开始给药(400 mg/kg,i.p.),共14 d。单独给药组给予VPA,同时用等量的生理盐水代替MPTP。盐水对照组仅给予等量的生理盐水。采用原位分子杂交方法观察BDNF的表达,并对检测部位恒定视野内BDNF的阳性细胞进行灰度扫描和统计学分析。结果显示:与盐水对照组相比,模型组、模型给药组和单独给药组小鼠纹状体、海马、皮质内BDNF的表达均增强。该结果提示,PD小鼠神经元内BDNF增多,可能有利于受损神经元的修复;丙戊酸盐可能通过促进BDNF的表达而保护神经元。     

Effects of aging, Parkinson's disease, and dopaminergic medication on response selection and control

We examined effects of short-term and long-term dopaminergic medication in Parkinson's disease on conflict monitoring or response selection processes. These processes were examined using event-related potentials (ERPs), while subjects performed a stimulus-response (S-R) compatibility task. An extended sample of young and elderly controls, Parkinson's disease patients with a medication history (PDs) and initially diagnosed, drug-naïve de novo PD patients (de novo PDs) were enrolled. Both PD groups were measured twice (on and off-medication or before and 8 weeks after medication onset).The results show that dopaminergic intervention selectively reduced the pathologically enhanced response selection in compatible S-R relations. This medication effect was already evident after short-term treatment, not differing from long-term treatment and performance in elderly controls. Contrary, age-related attenuations of the N2 in incompatible S-R relations, probably reflecting impaired conflict processing or response control, are unaffected by medication. The results suggest that compatible and incompatible S-R relations demand different neuronal mechanisms within the basal ganglia, as only the former are affected by agonizing the dopaminergic system.     

尼莫地平对帕金森病模型小鼠黑质多巴胺神经元的保护作用

目的:研究尼莫地平在1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)所致帕金森病(PD)模型小鼠中对黑质多巴胺能神经元的保护作用。方法:雄性健康C57BL/6N小鼠随机分为对照组,腹腔注射生理盐水;模型组,腹腔注射MPTP(25 mg/kg);尼莫地平治疗组,每次注射MPTP前3 h灌胃给予尼莫地平(15 mg/kg)。观察各组小鼠行为学变化,免疫组织化学和免疫蛋白印迹法观察中脑黑质酪氨酸羟化酶(TH)、前列腺素E2(PGE2)和肿瘤坏死因子α(TNF-α)的表达变化。结果:与对照组小鼠比较,MPTP模型组小鼠出现典型的PD症状,中脑黑质TH阳性神经元大量丢失,PGE2和TNF-α阳性细胞显著增多,蛋白水平明显升高;经尼莫地平治疗后,上述症状得到明显改善。结论:在MPTP所致PD小鼠模型中,尼莫地平通过抑制炎症因子PGE2和TNF-α的表达从而对多巴胺能神经元具有一定的保护作用。     

ATP13A2 protects dopaminergic neurons in Parkinson's disease: from biology to pathology

As a late endosomal/lysosomal transport protein of the P5-type, ATP13A2 is capable of removing the abnormal accumulation of α-synuclein, which maintains the homeostasis of metal ions and polyamines in the central nervous system. Furthermore, ATP13A2 regulates the normal function of several organelles such as lysosomes, endoplasmic reticulum (ER) and mitochondria, and maintains the normal physiological activity of neural cells. Especially, ATP13A2 protects dopaminergic (DA) neurons against environmental or genetically induced Parkinson's disease (PD). As we all know, PD is a neurodegenerative disease characterized by the loss of DA neurons in the substantia nigra pars compacta. An increasing number of studies have reported that the loss-of-function of ATP13A2 affects normal physiological processes of various organelles, leading to abnormalities and the death of DA neurons. Previous studies in our laboratory have also shown that ATP13A2 deletion intensifies the neuroinflammatory response induced by astrocytes, thus inducing DA neuronal injury. In addition to elucidating the normal structure and function of ATP13A2, this review summarized the pathological mechanisms of ATP13A2 mutations leading to PD in existing literature studies, deepening the understanding of ATP13A2 in the pathological process of PD and other related neurodegenerative diseases. This review provides inspiration for investigators to explore the essential regulatory role of ATP13A2 in PD in the future.     

Effect of dopaminergic substances on sleep/wakefulness in saline- and MPTP-treated mice

Sleep/wakefulness (S/W) disorders are frequent in Parkinson's disease (PD). The underlying causes have yet to be elucidated but dopaminergic neurodegenerative lesions seem to contribute to appearance of the disorders and anti-Parkinsonian medication is known to accentuate S/W problems. Hence, we reasoned that studying the acute effect of dopaminergic compounds on S/W in an animal model of PD might improve our knowledge of S/W regulation in the context of partial dopaminergic depletion. To this end, we tested the effect of levodopa (l-dopa), pergolide (a mixed D(2)/D(1) agonist) and lisuride (a D(2) agonist) on S/W recordings in MPTP-treated mice, in comparison with controls. Our results showed that dopaminergic compounds modify S/W amounts in both control and MPTP mice. Wakefulness amounts are greater in MPTP mice after l-dopa (50 mg kg(-1)) and lisuride (1 mg kg(-1)) injections compared with control mice. Moreover, the paradoxical sleep latency was significantly longer in MPTP mice after high-dose l-dopa administration. Our observations suggest that the actions of both l-dopa and lisuride on S/W differ slightly in MPTP mice relative to controls. Hence, MPTP-induced partial DA depletion may modulate the effect of dopaminergic compounds on S/W regulation.     

硫辛酸对LPS诱导的帕金森病小鼠黑质多巴胺能神经元损伤的影响

目的:探讨硫辛酸(LA)对脂多糖(LPS)诱导的帕金森病(PD)模型小鼠黑质多巴胺(DA)能神经元损伤的作用及机制。方法:10月龄雌性C57BL/6小鼠,随机分为生理盐水对照组、PD模型组和LA干预组。采用鼻腔滴入LPS制作PD小鼠模型,通过爬竿实验及酪氨酸羟化酶和p-NF-κB/p65的免疫组化技术观察LA对DA能神经元的保护作用。结果:LPS经鼻可以成功诱导小鼠PD模型,给予LA可明显恢复小鼠运动,减少黑质DA能神经元的丢失、抑制小胶质细胞及其NF-κB信号通路激活。结论:LA干预可缓解LPS经鼻诱导的小鼠PD行为学和病理学的改变,其作用机制可能与抑制脑内的炎症反应有关。     

丙戊酸钠对帕金森病模型小鼠多巴胺能神经元及脑源性神经营养因子表达的影响

帕金森病(Parkinson's disease,PD)是一种神经系统退行性疾病,主要表现为中脑黑质致密部多巴胺能神经元丧失,纹状体多巴胺含量下降,至今尚无有效的治疗手段.丙戊酸钠(Valproate, VPA)是临床上作为治疗双相精神障碍的主要药物.     

Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation

The role of astrocytes in microglia-induced neuronal death remains controversial. In this study, astrocytes and astrocyte-derived conditioned media (ACM) supported the survival of dopaminergic neurons, and the former was more effective than the latter. In the presence of astrocytes, low concentrations of LPS enhanced the survival of dopaminergic neurons, while high concentrations attenuated survival. LPS dramatically induced astrocytes to secrete IL-6 in a dose-dependent manner with no effect on secretion of GDNF. Neuron–astrocyte cultures had highest secretion of GDNF, followed by ACM-treated neuron-enriched cultures. After neuron–astrocyte cultures treated with IL-6-neutralizing antibody, both effects of the enhanced and attenuated survival of dopaminergic neurons were abolished. Our results indicate that astrocytes play a protective role in the LPS-induced damage of dopaminergic neurons in certain circumstances, and the interaction between astrocytes and dopaminergic neurons may enhance the protective effect of astrocytes. Suitable activation of astrocytes increases the protective effect while excessive activation attenuates it, and IL-6 might medicate this dual action. The underlying mechanisms related to the secretion of GDNF and proinflammatory factors warrant further investigation.