MPTP对褐鼠黑质NAPOR基因表达的影响

目的 研究腹腔注射1—甲基—4—苯基—1，2，3，6—四氢吡啶(MPTP)对褐鼠黑质神经母细胞瘤凋亡相关的RNA结合蛋白(NAPOR)基因表达的影响，为阐明帕金森病(PD)的分子机制提供线索。方法 通过腹腔注射MPTP建立褐鼠PD模型，采用逆转录PCR方法研究NAPOR在黑质的表达情况。结果 通过MPTP腹腔注射后褐鼠模型出现典型PD症状、黑质酪氨酸羟化酶表达水平明显降低验证PD模型成功，PD鼠黑质NAPOR基因表达明显上调。结论 NAPOR为RNA结合蛋白，其表达的模式与神经元凋亡密切相关。提示MPTP对黑质神经元的毒性可能通过改变NAPOR基因的表达，促进神经元凋亡而实现。     

MPTP对小鼠黑质及纹状体活性氧自由基代谢酶基因表达的影响

活性氧自由基是 1 甲基 4 苯基 1,2 ,3,6 四氢吡啶 (1 methyl 4 phenyl 1,2 ,3,6 tetrahydropyridine,MPTP)诱发多巴胺能神经元损伤的一种可能性因素。通过MPTP注射诱导C5 7BL小鼠神经元损伤 ,利用逆转录PCR方法检测铜锌超氧化物歧化酶 (CuZn SOD)、锰超氧化物歧化酶 (Mn SOD)以及谷胱甘肽过氧化物酶 (GSH PX)三种相关基因的表达变化。结果显示 ,在小鼠黑质与纹状体中 ,MPTP导致Mn SOD基因表达显著降低 ,而CuZn SOD与GSH PX基因的表达则没有明显变化。推测Mn SOD基因表达的变化可能与MPTP对线粒体的毒性作用有关。     

MPTP对小鼠纹状体多巴胺的影响

用高效液相色谱法检测Ｃ（５７）ＢＬ／６Ｊｏｌａ型老龄及幼龄鼠注射１－甲基－４－苯基－１、２、３、６四氢吡啶（ＭＰＴＰ）两周及两个月后纹状体多巴胺含量，发现幼龄鼠两个月后多巴胺含量由两周时的１．９±０．４μｇ／ｇ（以与对照组比较减少８２％）升高至５１．１±０．５μｇ／ｇ（与对照组比较减少５２％）；而老龄鼠则无明显改变。这说明龄鼠多巴胺神经元损害修复的功能减退，这可能是帕金森氏病多发于老年人的原因之一。     

基于氨基酸对含纤连蛋白域蛋白质亚细胞的定位预测*☆

背景:含FN域蛋白质在促进细胞迁移、黏附、生长、分化等方面发挥了重要功能,已被广泛应用于各种新型生物材料中。研究它们的亚细胞位置有利于它们的生物功能研究和新型生物材料开发。目的：实现含纤连蛋白域蛋白质的亚细胞定位预测。方法：从UniProt数据库中随机抽取80个人类含纤连蛋白域蛋白质。计算每个蛋白质的400种氨基酸对数量并组成400维向量。分别利用支持向量机和k最近邻法调用每个蛋白质的400维输入向量进行训练和测试。同时,利用jackknife检验法对测试结果进行检验。结果与结论：利用支持向量机法和k最近邻法法预测含纤连蛋白域蛋白质的亚细胞定位预测准确率分别为92.5%和95.0%。说明利用支持向量机和k最近邻法算法预测含纤连蛋白域蛋白质的亚细胞位置具有重要意义,有利于此类蛋白质的功能研究和新型生物材料的表面改造设计。     

MPTP与帕金森病小鼠模型的研究进展

自从人们发现 1 -甲基 -4 -苯基 -1、2、3、6-四氢吡啶 ( 1 -methy1 -4 -phenvl-1、2、3、6-te trahvdropvridine ,MPTP)能在人类诱发帕金森病以来 ,人们对MPTP的神经毒性作用作了大量研究 ,发现MPTP对脑黑质多巴胺能神经元有选择性的破坏作用 ,并且利用MPTP先后在灵长类动物猴和低等哺乳动物小鼠身上模拟成功了类似人类帕金森病病理变化及临床特征的动物模型。本文着重介绍MPTP选择性神经毒性作用和近年来帕金森病小鼠模型的有关研究工作。     

人脑胶质瘤尿激酶型纤溶酶原激活因子基因表达及意义

目的 探讨尿激酶型纤溶酶原激活因子（ｕＰＡ）基因在人脑胶质瘤中的表达及临床意义。方法 采用Ｎｏｒｔｈｅｒｎ印迹分子杂交和免疫组方法检测４３例人脑胶质瘤和５例正常脑组织ｕＰＡ ｍＲＮＡ的蛋白表达,并与临床床生物学参数进行综合分析。结果 上述组织或细胞均可表达２．５Ｋｂ的ｕＰＡ ｍＲＮＡ转录物,高级另胶质瘤较低级别和正常脑组织ｕＰＡ ｍＲＮＡ表达水平明显增高（Ｐ〈０．０１）,低级别胶质瘤与正常脑组织的     

MPTP对小鼠黑质及纹状体活性氧自由基代谢酶基因表达的影响

活性氧自由基是l-甲基4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine.MPTP)诱发多巴胺能神经元损伤的一种可能性因素.通过MPTP注射诱导C57BL小鼠神经元损伤,利用逆转录PCR方法检测铜锌超氧化物歧化酶(CuZn-SOD)、锰超氧化物歧化酶(Mn-SOD)以及谷胱甘肽过氧化物酶(GSH-PX)三种相关基因的表达变化.结果显示,在小鼠黑质与纹状体中,MPTP导致Mn-SOD基因表达显著降低,而CuZn-SOD与GSH-PX基因的表达则没有明显变化.推测Mn-SOD基因表达的变化可能与MPTP对线粒体的毒性作用有关.     

PLG和MPTP对纹状体多巴胺含量的影响

对小鼠应用较大剂量的MPTP后，其纹状体中DA，5－HT及其代谢产物的含量都降低。如在MPTP注射前先用PLG注入小鼠体内，虽然纹状体中DA含量也降低，但却显著高于单用MPTP组。结果表明PLG能部分地防止MPTP的神经毒性作用。其作用机制可能是PLG在纹状体直接或间接地结抗MPTP的神经毒性作用也可能阻止MPP^ 对多巴胺细胞内黑色素的亲和作用。     

MPTP小鼠黑质神经元细胞凋亡及Deprenyl抗凋亡作用的研究

目的：观察MPTP-Parkinsonism小鼠多巴胺能神经元细胞凋亡及Deprenyl抗凋亡作用;方法：应用TUNEL染色、TH、BCL－2免疫组化染色、TUNEL／TH双标染色和电镜技术观察多巴胺能神经元凋亡及Deprenyl抗凋亡作用;结果：模型鼠脑黑质内有多巴胺能神经元凋亡．而Deprenyl可阻止这种神经元凋亡;结论：1．模型中多巴胺能神经元的丢失可能是通过细胞凋亡机制实现的;2．Deprenyl可能部分阻止多巴胺能神经元凋亡。     

反义寡核苷酸阻断黑质多巴胺神经元多巴胺转运载体表达的大鼠对MPTP反应性的改变

研究表明多巴胺转运载体(dopamine transporter，DAT)是神经毒素1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)特异性载体，DAT选择性摄取MPTP进入黑质多巴胺神经元，造成神经元损伤。DAT表达水平受其基因多态性的影响。众多病例对照研究显示，多巴胺转运载体的多态性可能与PD遗传易患性有关，但尚有争议，也缺少直接的证据。我们采     

经MPTP处理的C57BL小鼠不同脑区多巴胺转运体与甘氨酸受体基因的表达

1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）对C57BL小鼠黑质多巴胺能神经元具有选择性的毒性作用,导致小鼠出现类似帕金森病的症状,利用抑制消减杂交技术分析MPTP对小鼠黑质基因表达的影响。获得了一系列差异表达基因片段,其中包括多巴胺转运体与甘氨酸受体两种与神经递质的功能与周转密切相关的基因,通过Northern杂交发现,这两种基因的表达在给药组小鼠的黑质中均下降,而在纹状体中多巴胺转运体基因的表达没有明显变化,甘氨酸受体基因的表达则有一定程度的增加。     

Selective alterations of gene expression in mice induced by MPTP

1-methyl-4-phenyl-1,2,4,6,-tetrahydropyridine (MPTP) is a selective neurotoxin that produces striatal dopamine depletion resulting in parkinsonism like symptoms in humans and is, therefore, used to generate animal models for Parkinson's disease (PD). In this study, C57BL/6N mice were treated with MPTP acutely (3x20 mg/kg, 2-hour interval, one day injection). Mice were then sacrificed 24 hours after the last injection and brain tissue was collected for analysis. Significant decrease of striatal dopamine (DA) and the metabolites (DOPAC, HVA) was observed after MPTP treatment. MPTP also reduced protein expression of tyrosine hydroxylase (TH) in the striatum. Real time RT-PCR was used to examine selective genes of the dopaminergic system in the substantia nigra. Our data demonstrated that MPTP significantly decreased gene expression of TH, dopamine transporter (DAT), and vesicle monoamine transporter (VMAT), coinciding with the pattern of dopamine concentration changes and protein expression after MPTP treatment. Although a significant decrease of DA metabolites was observed in striatum, there was no change in the expression of monoamine oxidases (MAO-A, MAO-B) or catechol O-methyltransferase (COMT), indicating that these changes might be simply a consequence of reduced monoamine levels. In addition, gene expression of alpha-synuclein was also decreased with MPTP treatment, but there was no change in beta-synuclein and parkin. This is the first study using real-time PCR to indicate that MPTP selectively alters gene expression and provides information for clinical studies in PD. Future studies will focus on gene expression of other pathways that may be affected by MPTP treatment and investigation of gene expression in specific cell types in vivo using LCM technology.     

利用消减抑制杂交技术构建MPTP诱导的C57BL小鼠全脑差异表达基因文库

本研究旨在构建1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的C57BL小鼠全脑差异表达基因文库,并获取差异表达的表达序列标签.通过MPTP腹腔注射建立C57BL小鼠帕金森病模型.利用消减抑制杂交技术建立对照组与实验组差异表达基因文库.自两文库中各随机挑选20个克隆,经反Northern杂交证实其中7个克隆为阳性.同时对上述40个克隆进行序列测定,并与已知序列基因库进行同源性比较,其中26个新基因片段向Genbank申请了序列号.     

Differential effects of advancing age on neurotransmitter cell loss in the substantia nigra and striatum of C57BL/6N mice

The present study was carried out to examine the extrapyramidal motor system of C57BL/6N mice for age-related cell loss in cholinergic neurons of the striatum (ST) and dopaminergic (DA) neurons of the substantia nigra (SN). Immunocytochemistry using antibodies against tyrosine hydroxylase (TH) or choline acetyltransferase (CAT) were used to identify DA or cholinergic neurons of the SN and ST in 6 age groups of young (3 months), middle (6, 10, 20 months) and old (25, 30 months) aged mice. We found that while there was a small decline (11%) in the total number of DA neurons of the SN with age, this decrease did not reach statistical significance. In contrast, the total number of cholinergic neurons of the ST significantly decreased between 25 and 30 months of age with the largest cell loss (38%) found in the rostral ST. In addition, the loss of cholinergic neurons in 30-month-old mice was paralleled by a decline in the mean cross-sectional area of the cell soma and nucleus of remaining cholinergic neurons. These data suggest that advancing age has a differential effect on neurotransmitter neurons of the SN and ST and supports the notion that cell loss is not an inevitable characteristic of senescence but is brain region- and cell type-specific. In addition, these data are consistent with the hypothesis that the proliferation of striatal dendrites described previously in aged C57BL/6N mice may result, in part, from a compensatory growth of these processes secondary to age-related cell loss of striatal neurons.     

利用消减抑制杂交技术构建MPTP诱导的C57BL小鼠全脑差异表达基因文库

本研究旨在构建 1 甲基 4 苯基 1,2 ,3,6 四氢吡啶 (MPTP)诱导的C5 7BL小鼠全脑差异表达基因文库 ,并获取差异表达的表达序列标签。通过MPTP腹腔注射建立C5 7BL小鼠帕金森病模型。利用消减抑制杂交技术建立对照组与实验组差异表达基因文库。自两文库中各随机挑选 2 0个克隆 ,经反Northern杂交证实其中 7个克隆为阳性。同时对上述 4 0个克隆进行序列测定 ,并与已知序列基因库进行同源性比较 ,其中 2 6个新基因片段向Genbank申请了序列号。     

Maternal voluntary drinking in C57BL/6J mice: advancing a model for fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) remain the most common preventable cause of behavioural abnormalities and cognitive deficits, yet little is known about the biological mechanisms involved in FASD pathology. Maternal voluntary ethanol consumption in mice may be a useful model for establishing the biological basis of moderate ethanol exposure phenotypes, which make up the majority of FASD cases. We have employed a two-bottle choice paradigm of maternal ethanol consumption throughout gestation and the early postnatal period in C57BL/6J mice. We assessed the efficacy of this model to produce a range of FASD-relevant phenotypes and evaluated gene expression changes in the adult offspring. Results showed stable maternal consumption and lack of maternal care differences between ethanol-consuming and water-only dams. Ethanol-exposed offspring showed delays in neonatal reflex and coordination development. Further, ethanol-exposed adolescent mice showed decreased activity in a novel environment that appeared to be the result of novelty-induced anxiety, and acquisition learning deficits. Evaluation of the neurotransmitter-associated genes Gabra6, Glra1, and Grin2c revealed significant down-regulation of Glra1 and Grin2c in the brains of ethanol-exposed young adult males. These results suggest that this model is able to produce a range of behavioural phenotypes consistent with prenatal ethanol exposure and may be used to evaluate resulting long-term genetic changes. Given the range of genetic resources available for inbred mouse strains, the model described here may prove to be a useful tool in evaluating the molecular basis of FASD.     

C57BL小鼠survivin基因miRNA表达载体的构建*★

背景：Survivin在多种肿瘤组织中的高表达，具有调节细胞增殖分裂和强大的抗凋亡功能。 目的：利用RNA干扰技术，构建具有特异性阻断C57BL小鼠survivin基因的微小RNA(micro RNA，miRNA)表达载体。 设计、时间及地点：单一样本观察，于2008-06/11在重庆医科大学附属第一医院神经内科实验中心完成。 材料：环形pcDNA™6.2-GW/EmGFPmiR和BLOCK-iT™ Pol II miR RNA干扰 Expression Vector Kit with EmGFP为Invitrogen公司产品；DH5a大肠杆菌为实验室保存；xho I和BamH I酶、壮观霉素均为上海生工生物工程有限公司产品。 方法：应用设计软件在C57BL小鼠survivin基因的mRNA上寻找特异性的短核苷酸序列，并设计合成4对寡核苷酸序列，经退火后形成双链DNA片段，采用基因克隆技术，将其克隆到pcDNA™6.2-GW/EmGFPmiR的载体中，转化DH5a大肠杆菌，挑单菌落种于含有壮观霉素LB液体培养基中，提取质粒。 主要观察指标：应用测序法和琼脂糖电泳检测对重组体进行鉴定。 结果：测序结果显示插入片段与线性载体连接正确，无碱基突变、缺失、插入等异常。双酶切处理pcDNA™6.2-GW/EmGFP-miR重组质粒结果显示片段大小与预期相符。 结论：实验结果表明成功构建了C57BL小鼠survivin基因的微小RNA表达载体。