家族性帕金森病患者parkin基因缺失突变的初步研究

目的：探讨中国家族性帕金森病(parkinson’s disease，PD)患者parkin基因第3～7外显子是否存在缺失突变，及其与该病临床特点的关系。方法：采集6例无血缘相关的家族性PD患者外周血液，提取DNA，通过PCR扩增、琼脂糖凝胶电泳鉴定parkin基因第3～7外显子缺失突变，并结合临床资料分析。结果：6例无血缘相关的家族性PD患者中，发现1例有第5外显子缺失，其遗传模式呈常染色体隐性遗传，起病年龄60岁，临床表现为震颤、僵直和运动迟缓，但无异动症。第3，4，6，7外显子未发现缺失突变。结论：中国家族性PD患者中存在parkin基因第5外显子缺失突变改变。     

应用培高利特单药治疗早期帕金森病:随机、双盲、对照实验

目的:观察应用培高利特单药治疗早期帕金森病的疗效,为合理应用多巴胺受体激动剂干预帕金森病提供依据。方法:60例首次接受治疗的早期帕金森病患者为2002-10/2004-10湘雅医院收治,将患者随机分为两组:培高利特组、多巴丝肼组,分别于治疗前、治疗1,3及6个月后进行帕金森病评定量表评分、改良Hoehn-Yahr分级评分及临床疗效观察。结果:治疗3个月后,培高利特组改良Hoehn-Yahr分级评分与多巴丝肼组相比无统计学意义(P>0.05);治疗6个月后,培高利特组帕金森病评定量表评分与多巴丝肼组相比差异无显著性意义(P>0.05)。治疗6个月后,培高利特组的临床疗效与多巴丝肼组相比差异无显著性意义(P>0.05)。副反应观察发现,培高利特组患者副反应的发生率低于多巴丝肼组(30%比50%,P<0.01),其中培高利特组患者出现幻觉的比例稍高于多巴丝肼组,恶心、失眠、便秘、直立性低血压等副反应的发生率均低于多巴丝肼组(P<0.01)。结论:应用培高利特单药治疗早期帕金森病6个月后疗效与多巴丝肼相似,且副反应少。     

烟碱对大鼠脑组织多巴胺D1、D2受体mRNA表达的影响

研究表明 ,吸烟可降低帕金森病 (ＰＤ)的发病率〔1〕。烟碱可能通过诱导大鼠脑内神经营养因子 (ＢＤＮＦ)的合成及抑制羟自由基的产生等〔2〕,而对多巴胺神经元具有保护效应。本课题于 2 0 0 0年 10月至 2 0 0 1年 4月进行 ,旨在研究烟碱对大鼠脑组织多巴胺受体 (Ｄ1、Ｄ2 受体 )ｍＲＮＡ达的影响。　　一、材料与方法　　 1 实验动物和分组 :2 4只健康雄性清洁级ＳＤ大鼠 ,10周龄 ,重 180～ 2 0 0ｇ ,由中南大学湘雅医学院实验动物学部提供。动物随机分为 2组 :生理盐水对照组 (12只 ) ;烟碱实验组 (12只 )。分别给予生理盐水、烟碱 (美国…     

尼古T对MPTP-PD小鼠纹状体NOS活性的影响

为观察帕金森病(Parkinson disease,PD)模型小鼠脑组织中一氧化氮合酶(NOS)活性的变化,研究尼古丁在PD中的可能作用机制.本实验采用1-甲基4-苯基-l,2,3,6-四氢吡啶(MPTP)建立C57BL小鼠PD模型,分别应用比色分析、高效液相色谱-电化学以及免疫组织化学检测MPTP和尼古丁对C57BL小鼠纹状体NOS活性,多巴胺(DA)、二羟基苯乙酸(DOPAC)、高香草酸(HVA)水平及酪氨酸羟化酶(TH)、神经元型一氧化氮合酶(nNOS)免疫组化的影响.结果发现尼古丁能明显抑制MPrP引起的NOS活性增加(每mg蛋白质分别为15.63IU±1.5 Iu和13.IU±0.89 Iu,P<0.01)及nNOS阳性神经元的数量(分别为68.61±3.84和39.26±2.64,P<0.01),并能明显减轻MPTP引起的小鼠纹状体DA(每g脑组织中分别为0.73ug±0.28ug和1.40ug±O.19ug,P<0.01)、DOPAC(每g脑组织中分别为0.4lug±0.13ug和0.90ug±0.09ug,P<0.01)、HVA(分别为0.31ug±0.07ug和O.47ug±0.19ug,P<0.01)的降低及TH阳性神经纤维的损害.因此认为,尼古丁可能通过抑制nNOS的活性而对MPTP的神经毒性具有保护作用.     

应用SYBR GreenⅠ实时荧光定量聚合酶链反应检测常染色体隐性遗传早发性帕金森综合征的parkin基因外显子重排突变

目的 建立应用SYBR GreenⅠ实时荧光定量聚合酶链反应(Real-time PCR,RT-PCR)检测parkin基因外显子重排突变的技术平台,应用该技术对常染色体隐性遗传早发型帕金森综合征(autosomal recessive early-onset parkinsonism,AREP) 家系进行parkin基因外显子重排突变分析.方法 应用SYBR GreenⅠRT-PCR技术对32个中国AREP家系进行parkin基因外显子重排突变分析.结果 14个家系先证者存在parkin基因外显子重排突变,其中3个为纯合缺失突变、3个为复杂杂合缺失突变和8个杂合缺失突变,未发现外显子重复突变,突变主要累及第2～4号外显子.结论 建立了应用SYBR GreenⅠRT-PCR技术检测parkin基因外显子重排突变的基因检测平台;中国AREP 家系的parkin基因外显子重排突变频率为43.8%,与国外报道相似.     

RNA干扰技术阻断α-synuclein过表达诱导的细胞凋亡

BACKGROUND： Overexpression of α-synuclein can induce cell apoptosis. RNA interference （RNAi） may block specific gene function and cause gene silencing. OBJECTIVE： To construct a specific and effective RNAi plasmid for the α-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein.
DESIGN, TIME AND SETTING： A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008.
MATERIALS： HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA.
METHODS： Four target sites were used to construct hairpin RNA pBSHH1 vectors - pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4 - which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows： transfection of blank plasmid （blank control group）, transfection of α-synuclein-pEGFP and RNAi negative vector （negative control group）, and transfection of α-synuclein-pEGFP and pSYNi-1 （transfection group）. Cells in all groups were transfected with Lipofectamine 2000 for 48 hours.
MAIN OUTCOME MEASURES： Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry.
RESULTS： α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups （P 〈 0.05）. The expressions were partially decreased in the pSYNi-2 group, but there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased （P 〈 0.05）. In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased （P 〈 0.05）.
CONCLUSION： pSYNi-1 can inhibit α-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein.     

野生型DJ-1 与A39S 突变型DJ-1 表达的单克隆细胞株基因表达谱分析

目的：探讨DJ-1 基因A39S 突变在帕金森病发病过程中的作用机制,以及其是否可能通过转录调控活性
导致相关基因表达异常。方法：建立能够稳定表达空载体和野生型DJ-1 蛋白,以及A39S 突变型DJ-1 蛋白的HEK293
单克隆细胞株。利用基因芯片技术对不同组别细胞株进行差异性表达基因筛选。结果：与空载体组比较,野生型
DJ-1 组有14 个基因表达上调,28 个基因表达下调;A39S 突变型DJ-1 组有6 个基因表达上调;2 个基因表达下调;
A39S 突变型DJ-1 组与野生型型DJ-1 组比较发现只有1 个基因表达下调。具有表达差异性的基因分别参与信号转导、
基因转录调控、细胞周期、细胞凋亡、氧化应激等生物学过程。结论：A39S 突变型DJ-1 蛋白可能通过直接或间接方
式对这些差异基因进行表达调控影响这些通路的正常功能,而参与帕金森病的发病。     

动态脑电图在癫痫诊断及鉴别诊断中的价值

目的研究24 h动态脑电图(AEEG)在癫(疒间)的诊断及鉴别诊断中的价值.方法对761例临床拟诊为癫(疒间)及1327例可疑癫(疒间)患者的AEEG资料进行回顾性分析.结果两组AEEG与常规脑电图检查(疒间)样放电检出率比较差异有极显著性(均P＜0.005).230例患者通过AEEG监测证实为癫(疒间)发作.93例患者AEEG监测后发作类型得到了修正.结论 AEEG有助于癫(疒间)的诊断及鉴别诊断.     

远端型遗传性运动神经病的小分子热休克蛋白22、27基因突变分析

远端型遗传性运动神经病(distalhereditarymotor neuropathy,dHMN)是一组由于脊髓前角运动神经元退行性变引起对称性的肌无力和肌萎缩,常成年起病,病程一般缓慢发展。到目前为止,dHMN已定位9型,其中5型疾病基因已被克隆,HSP22(heat shockprotein22)基因和HSP27(heatshockprotei