Nurr1在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响

目的 :观察MN9D细胞中Nurr1的表达和分布及Nurr1过表达对酪氨酸羟化酶的影响。 方法 :应用高压液相色谱、免疫细胞化学、转基因、Westernblot和Northernblot等方法检测了MN9D细胞中多巴胺及其代谢物的水平和Nurr1蛋白在细胞中的分布 ,建立了过表达Nurr1的细胞株并观察了Nurr1过表达对TH基因的影响。结果 :MN9D细胞裂解液中含有大量多巴胺及其代谢物 ;Nurr1蛋白主要分布在MN9D细胞的胞质中 ,尤以核周最多 ;MN9D细胞中有Nurr1的mRNA存在和蛋白质的表达 ;过表达Nurr1的MN9D细胞A1株酪氨酸羟化酶表达增高。结论 :Nurr1与多巴胺能神经元发育有关 ,有可能用于帕金森病的基因治疗。     

Nurrl在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响

目的：观察MN9D细胞中Nurrl的表达和分布及Nurrl过表达对酪氨酸羟化酶的影响。方法：应用高压液相色谱、免疫细胞化学、转基因、Westernblot和Northemblot等方法检测了MN9D细胞中多巴胺及其代谢物的水平和Nurr1蛋白在细胞中的分布，建立了过表达Nurr1的细胞株并观察了Nurr1过表达对TH基因的影响。结果：MN9D细胞裂解液中含有大量多巴胺及其代谢物；Nurr1蛋白主要分布在MN9D细胞的胞质中，尤以核周最多；MN9D细胞中有Nurr1的mRNA存在和蛋白质的表达；过表达Nurr1的MN9D细胞A1株酪氨酸羟化酶表达增高。结论：Nurr1与多巴胺能神经元发育有关。有可能用于帕金森病的基因治疗。     

核受体相关因子1 shRNA表达载体的构建及其对多巴胺能细胞内源基因的干扰作用

目的 构建小鼠核受体相关因子1（Nurr1）shRNA的真核表达载体,并检测其对多巴胺能细胞内源基因的干扰作用.方法 构建两个针对Nurr1的RNA干扰重组表达载体,分别命名为pSC-N1和pSC-N2.经测序确认后,转染多巴胺能神经前体细胞系MN9D细胞株,以实时荧光定量PCR以及Western blot方法检测转染后Nurr1 mRNA和蛋白的表达.结果 测序鉴定证实合成的siRNA基因序列正确并已被准确克隆入pSilenCircle载体.pSC-N1和pSC-N2可特异性抑制MN9D细胞中Nurr1 mRNA的表达,下降率分别为62.3%和45.6%;Nurr1蛋白的表达亦明显下调,其下降率分别为57.4%和72.0%,而对照质粒对其无抑制作用.结论 成功构建了能特异性抑制多巴胺能细胞内源Nurr1表达的shRNA表达载体,为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础.     

GFP-Nurr1基因修饰神经干细胞的建立及过表达Nurr1对神经干细胞向多巴胺神经元分化的影响

目的利用慢病毒载体建立GFP-Nurr1基因修饰的原代神经干细胞(NSCs)模型并观察Nurr1过表达后NSCs向多巴胺神经元的分化影响。方法利用基因重组构建pLenO-DCE-Nurr1慢病毒载体,用慢病毒转染第三代NSCs,转染72 h后荧光检测转染效果;设置空白对照组、空载体组及DCE-Nurr1组,分别用Western blot及PCR检测Nurr1的表达差异;并将转染后的NSCs分化培养7 d后分别用免疫细胞化学检测、Western blot及PCR检测酪氨酸羟化酶(TH)的表达。结果慢病毒转染NSCs 72 h后,转染率可达90%,与对照组相比DCE-Nurr1组高表达Nurr1。经慢病毒载体感染后的NSCs仍具备分化潜能,分化培养后发现DCE-Nurr1组分化的神经细胞中TH阳性细胞分化率90.60%,对照组为21.2%。结论慢病毒载体可高效转染NSCs过表达Nurr1;Nurr1基因过表达可以促进中脑腹侧来源NSCs向TH阳性多巴胺能神经元方向分化。     

Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究

目的探讨联合过表达核受体相关因子1(Nurr1)基因的小胶质细胞(MG)和神经干细胞(NSC)共培养对神经干细胞向多巴胺神经元分化的影响。方法原代培养SD大鼠神经干细胞和小胶质细胞,并过表达Nurr1基因。CCK-8法检测Nurr1过表达对神经干细胞以及小胶质细胞活率的影响。Transwell系统共培养神经干细胞和小胶质细胞,实验分为NSC组、NSC+MG组和N(NSC+MG)组。ELISA检测共培养后第3天、第6天和第9天各组脑源性神经营养因子(BDNF)、血小板源性神经营养因子(PDNF)和胶质细胞源性神经营养因子(GDNF)表达变化;RT-PCR和Western Blot检测各组第9天酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)DAT和Nurr1的表达变化;细胞免疫荧光鉴定神经干细胞的分化,并对TH和DAT阳性细胞计数,计算各组神经干细胞向多巴胺神经元的分化效率。结果原代培养小胶质细胞以及神经干细胞并成功过表达Nurr1基因。CCK-8法检测结果表明,Nurr1过表达对神经干细胞以及小胶质细胞活率无明显影响。ELISA检测结果表明,N(NSC+MG)组在不同时间点神经营养因子(BDNF、PDNF和GDNF)表达量明显高于其他各组(P0.05)。RT-PCR和Westen Blot检测结果表明,N(NSC+MG)组TH、DAT和Nurr1的表达水平明显高于其他各组(P0.05)。细胞免疫荧光鉴定结果表明,N(NSC+MG)组TH阳性细胞率明显高于其他各组(P0.05)。结论Nurr1基因可促进神经干细胞和小胶质细胞共培养系统神经营养因子的分泌。过表达Nurr1基因的神经干细胞和小胶质细胞共培养可促进神经干细胞向多巴胺神经元的分化。     

Nurr1基因转染人脐血间充质干细胞诱导分化为多巴胺能神经元的实验研究

目的探讨外源性Nurrl基因修饰的人脐血间充质干细胞在基因重组成纤维细胞生长因子-8(FGF8)和音猬因子(Shh)诱导下体外分化为多巴胺能神经元的情况。方法间充质干细胞被随机分为A组(对照组)、B组(Nurrl组)、C组(FGF8+Shh组)及D组(Nurrl+FGF8+Shh),采用脂质体法将pcDNA3.1-Ntrr1转染B、D组间充质干细胞,Western blot观察Nurr1基因表达情况,并在神经元条件培养液中进行增殖培养和诱导分化,间接免疫荧光染色法鉴定细胞性质,高效液相色谱法测定多巴胺含量。结果Western blot结果显示转染后Nurr1蛋白表达明显增高。A组和B组未发现酪氨酸羟化酶(TH)阳性细胞,而C组和D组TH阳性细胞分别为10．12％±2．65％及25．36％±3．13％,多巴胺含量分别为(43．6±2．1)nmol／L及(83．2±3．5)nmol／L,差异均有显著性意义(P< 0．01)。结论人脐血间充质干细胞在FGF8和Shh诱导下可以分化为多巴胺能神经元,外源性Nurr1基因修饰后,分化为多巴胺能神经元的数量增加。     

帕金森病大鼠黑质NurrlmRNA表达的动态变化

目的　探讨帕金森病大鼠黑质核内受体相关因子 1(Nurr1)mRNA表达的动态变化。方法　通过脑立体定位注射 6 羟基多巴胺 (6 OHDA)的方法建立大鼠帕金森病 (Parkinson’sdisease,PD)模型 ,采用HE染色 ,酪氨酸羟化酶 (TH)免疫组织化学染色、原位杂交技术 ,选择 6 OHDA注射术后 1d、3d、5d、7d、2 1d为研究时点 ,观察大鼠PD模型形成过程中黑质TH 多巴胺细胞数量及Nurr1mRNA表达的改变。结果　与健侧比较 ,注射 6 OHDA 5d组损毁侧黑质TH 细胞显著减少 (P <0 .0 1) ,2 1d时仅为健侧的 15 %且出现明显的旋转行为 ;同时 ,注射 6 OHDA 1d组损毁侧黑质Nurr1mRNA表达即开始下降 ,且以 3d组最为显著 (P <0 .0 1) ,7d以后各组完全消失。结论　本实验研究结果表明 ,6 OHDA能下调大鼠黑质Nurr1mRNA的表达早于诱导多巴胺细胞的死亡     

小胶质细胞介导帕金森病模型小鼠的炎症损伤*

背景：研究表明帕金森病的黑质纹状体存在小胶质细胞的激活,但其释放炎症因子在帕金森病发病过程中的作用尚不明确。 目的：观察小胶质细胞激活所释放的两种炎症因子整合素α及肿瘤坏死因子α在小鼠黑质部位的蛋白表达及酪氨酸羟化酶的蛋白表达。 方法：将C57BL/6小鼠经腹腔注射百草枯(10 mg/kg),设为帕金森病模型组,并设置对照组,观察两组小鼠行为活动。用高效液相法测定小鼠黑质纹状体多巴胺的含量。采用免疫组织化学法检测黑质部位酪氨酸羟化酶、整合素α及肿瘤坏死因子α蛋白表达。 结果与结论：百草枯可造成帕金森病模型组小鼠运动减少,并伴有运动迟缓、震颤、探嗅、竖毛及尾巴硬类似于帕金森病样的行为表现,并且脑内黑质纹状体多巴胺含量降低(P < 0.05),酪氨酸羟化酶蛋白表达降低(P < 0.05),整合素α和肿瘤坏死因子α蛋白表达增加(P < 0.05),肿瘤坏死因子α在模型组小鼠黑质纹状体有阳性表达,且主要分布在小胶质细胞上。结果表明小胶质细胞介导的炎症损伤参与了帕金森多巴胺能神经元的损害过程。     

中脑腹侧区细胞移植对帕金森病鼠纹状体多巴胺受体敏感性的影响

大鼠纹状体中多巴胺受体介导的c-fos基因的表达与多巴胺D1受体的超敏现象有关。本实验将鼠胚胎中脑腹侧区细胞植入帕金森病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,同时取相邻切片进行酪氨酸羟化酶检测。结果经图像分析发现胚胎中脑腹侧区移植,能显著减少移植侧纹状体中c-fos的表达量,说明胚胎中脑腹侧区细胞移植能够纠正多巴胺受体的超敏现象。     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.     

Summary of legislation of 1935 of interest to the department of Mental Hygiene

Psychiatric Quarterly -