耐药性癫癎患者脑组织线粒体mMDH NDUFC2基因表达异常

目的研究耐药性癫癎患者脑细胞线粒体mMDH、NDUFC2表达,探讨其在耐药性癫癎形成中的作用.方法分别提取48例耐药性癫癎患者、8例非耐药性癫癎患者及8例对照组脑组织标本的总RNA后,用基因芯片对其进行扫描,随后用荧光定量PCR技术进行验证.结果发现与线粒体功能有关的基因mMDH、NDUFC2在耐药性癫癎中显著下调,荧光定量RT-PCR验证结果与基因芯片一致.结论脑细胞线粒体基因mMDH、NDUFC2表达异常可能通过能量代谢及神经元坏死参与了耐药性癫癎的形成.     

基因芯片检测难治性癫脑组织中sh3gl2的表达

目的：运用基因芯片和RT-PCR技术检测难治性癫脑组织中sh3gl2 mRNA表达,从分子水平探讨难治性癫可能的发病机制。方法：在应用基因芯片对难治性癫患者手术切除的颞叶组织与对照组行基因表达谱分析研究的基础上,筛选出目的基因后用RT-PCR对芯片扫描结果进行验证。结果：候选基因sh3gl2在难治性癫患者脑部颞叶组织中出现高表达,与对照组相比差异有显著统计学意义（P〈0.01）;RT-PCR结果与芯片结果一致。结论：sh3gl2在难治性癫颞叶皮质中的表达增加,提示了其可能是难治性癫发生发展中的一个重要因素。     

5-HT2C受体在癫患者脑组织的表达

目的研究5-羟色胺2C受体(5-HT2C受体)在癫患者脑组织的表达,探索其临床意义。方法用免疫组化方法检测46例癫患者术后脑组织5-HT2C受体的表达,研究比较不同部位不同病程脑组织表达。结果癫患者额叶脑组织5-HT2C受体的含量较颞叶海马明显增高,长病程组表达低于短病程组。结论 5-HT2C受体可能参与了癫的发病机制,改变5-HT2C受体的表达有望成为癫新的治疗靶点。     

37例女性癫患者月经期癫发作的临床分析

目的探讨女性癫患者月经期癫发作的特点、原因及治疗经验。方法回顾性分析2007—2012年我科收治的37例月经期癫发作的病例。结果女性癫患者月经期癫发作加重的主要表现为癫的发作频率的增加,月经期癫发作加重与女性月经期体内的雌激素及孕激素水平、大脑皮质和海马神经细胞内雌激素及孕激素受体水平以及女性月经期AEDs药物清除能力增强等因素有关。结论增加AEDs的用药次数、用量或联合用药治疗女性癫患者月经期癫发作安全有效。     

难治性癫患者颞叶GFAP表达的变化

目的观察难治性癫患者手术切除的颞叶脑组织中GFAP表达的变化,以探讨颞叶癫的发病机制。方法以12例难治性癫患者为实验组,2例意外死亡的健康人为对照组,采用免疫组织化学方法观察2组颞叶脑组织中GFAP表达的变化。结果与对照组相比,实验组颞叶GFAP的表达显著升高(P<0.05)。结论反应性胶质细胞增生参与了癫发生的病理过程,GFAP的表达上调可能是难治性癫发病的分子学机制之一。     

癫患者抗氧自由基治疗后脑电图及发作频数临床观察

目的观察抗氧自由基治疗对癫患者脑电图和发作频数的影响。方法选择2009-01—2012-05于我院神经内科就诊并被诊断为癫患者63例,将所有入选患者按奇数、偶数分为对照组31例和治疗组32例,对照组采用基础抗癫治疗,治疗组在对照组基础上给予抗氧自由基治疗(维生素C、E)。治疗3个月后进行疗效对比。结果治疗组显效21例,有效5例,改善4例,总有效率93.7%;对照组显效15例,有效3例,改善2例,总有效率64.5%,2组总有效率比较差异有统计学意义(P<0.01)。结论抗自由基治疗能够增强抗癫基础治疗的效果,减少癫发作次数和放电频数,疗效显著,可在临床广泛使用。     

癫动态脑电图与CT异常的关系分析

目的探讨AEEG和CT在癫诊断、鉴别诊断和病因诊断中的作用。方法本文分析2005-01～2006-12,采用GE-1800型CT检查的91例CT异常的癫患者,与SOLARROVER-8型AEEG检查的结果。结果AEEG正常27例(29.7%),异常64例(70.3%)。其中样波32例,占异常的50.0%;非特异性异常32例(50.0%)。临床发作14例占异常的21.9%。结论AEEG的临床应用,大大提高了癫脑电图的阳性率。特别对无明显临床发作表现和先兆指征的可疑性癫患者进行诊断和分类。CT广泛应用使脑部疾病引起的癫阳性率大为提高,降低了原发性癫的诊断率,已成为癫病因研究的重要手段,它能寻找癫最可能和最重要的潜在病因。CT与脑电图二者结合在癫病因诊断及定位诊断中起到相辅相成的作用,对癫的治疗,预后帮助很大。     

难治性癫患者外周血中MDR1基因的表达及临床意义

目的研究难治性癫患者外周血中多药耐药1(MDR1)基因的表达以及抗癫药物和性发作在难治性癫多药耐药发生中的作用。方法采用逆转录聚合酶链反应(RT-PCR)半定量检测120例研究对象外周血中MDR1基因mRNA的表达。根据析因设计分性发作和抗癫药物两个因素,共分为A组(难治性癫组)、B组(癫治疗有效组)、C组(性发作未用药组)和D组(健康正常对照组),各30例。结果4组的外周血中MDR1基因的表达水平明显不同(F=4.456,P=0.005),其中性发作引起MDR1mRNA的表达明显增高(F=10.005,P=0.002),抗癫药物的作用则不明显(F=0.919,P=0.340),抗癫药物与性发作两者之间没有交互作用(F=2.445,P=0.121)。结论难治性癫患者外周血中MDR1基因mRNA的表达上调是性发作而非使用抗癫药物的结果,外周血中MDR1基因mRNA表达水平的监测可以作为评价癫耐药的一项指标。     

老年性脑出血患者继发癫36例临床分析

脑出血是一种常见的急性脑血管病,严重危害人民的健康,是引起中老年人癫的常见原因。近年来国内外对卒中后癫的研究越来越重视,为进一步探讨脑出血后癫的临床表现和预后,我们对我院2000-01～2005-12确诊的老年性脑出血患者377例,其中36例继发癫,报告如下。1临床资料1.1一般资料本组男20例,女16例,年龄60～101岁,平均71岁,均符合全国第四届脑血管病学术会议制定的脑血管疾病诊断标准,并经头颅CT证实,其中脑出血33例,蛛网膜下腔出血3例。脑出血后癫的诊断标准:所有病例均于本次脑出血后出现癫发作,既往无类似发作,且可除外去大脑…     

癫痫患者脑组织PSD-93 mRNA表达上调

目的研究癫痫患者脑组织中突触后密度-93(PSD-93)mRNA的表达,探讨其在癫痫形成中的作用。方法将56例癫痫患者分成耐药和非耐药两组,首先用基因芯片对其术后标本进行扫描,并与对照组比较,随后对目标基因PSD-93用逆转录聚合酶链反应(RT-PCR)技术进行验证。结果基因芯片检测发现与依赖N-甲基-D-天门冬氨酸受体(NMDARs)-一氧化氮(NO)信号有关的基因PSD-93在癫痫患者中表达上调,RT-PCR实验结果与基因芯片一致。RT-PCR电泳图的灰度值均数在对照组为23.577、非难治性癫痫组为56.931、难治性癫痫组为51.607,非难治性、难治性癫痫组与正常对照组灰度的比值中位数分别为2.415、2.189(P<0.05),而非难治性与难治性癫痫组灰度值比值为1.103(P>0.05)。结论脑细胞膜相关鸟苷酸激酶家族(MAGUK)蛋白信号转导复合体的分子接头蛋白基因PSD-93mRNA表达增加,其可能通过信号传导异常及神经元坏死参与了癫痫的形成。     

cDNA芯片检测耐/非耐苯妥英钠癫痫鼠脑线粒体基因的差异表达

目的：通过了解耐/非耐苯妥英钠（PHT）癫痫鼠与人类同源线粒体基因的差异表达来探索难治性癫痫的分子病理机制。方法：建立PHT耐药和非耐按照癫痫鼠模型，取脑组织常规抽提Mr-NA，逆转录生成cDNA并标记后，与含有4096条人类已知基因的cDNA表达谱芯片杂交，检测线粒体内37个基因及线粒体外相关基因在两者间的差异表达。结果：发现耐PHT鼠脑线粒体37条基因中有12条基因表达异常。结论：脑细胞线粒体基因表达异常可能是难治性癫痫的分子病理基础，能量代谢障碍和神经元凋亡在难治性癫痫形成，尤其是后期的发生和发展起着重要作用。     

内侧颞叶癫病人脑组织GABRG2基因变异研究

目的探讨GABRG2基因变异与内侧颢叶癫癎的关系.方法运用RT-PCR方法测定内侧颞叶癫癎致癎灶和其他类型癫癎病人手术切除脑组织内的GABRG2基因mRNA序列.结果全组共发现3处单核苷酸多态性,其电内侧颞叶癫癎组245 G→A出现频率与对照组存在统计学差异.结论GABRG2基因突变极有可能在内侧颞叶癫癎的发病中起重要作用.     

Safe and effective use of the ketogenic diet in children with epilepsy and mitochondrial respiratory chain complex defects

PURPOSE: To evaluate the clinical efficacy and safety of the ketogenic diet (KD) for patients with intractable childhood epilepsy and mitochondrial respiratory chain (RC) complex defects. METHODS: A retrospective analysis evaluated outcomes in 14 children with intractable epilepsy and RC complex defects who were treated with the classic KD involving a 4:1 lipid to nonlipid ratio (% by weight), but without an initial fast and fluid restriction. Outcome measures included seizure frequency, electroencephalography (EEG) findings, the number of antiepileptic drugs, and adverse reactions. RESULTS: Of the 14 patients, 9 had Complex I defects, 1 had a Complex II defect, 3 had Complex IV defects, and 1 had combined Complex I and IV defects. Two patients with Complex IV defects showed clinical progress compatible with the Leigh disease. The epileptic diagnoses were as follows: 5 patients were diagnosed with infantile spasms, 4 with the Lennox-Gastaut syndrome, 1 with the Landau-Kleffner syndrome, 1 with nonspecific generalized seizure disorder, and 3 with partial seizure disorder. The study found that 7 patients became seizure-free after commencing the KD, three of whom successfully completed the diet without relapse. One patient with a greater than 90% seizure reduction, and 2 patients with seizure reductions between 50% and 90%, remained on the diet. Four patients, including two diagnosed with the Leigh disease, did not show any favorable responses to the diet or ceased the diet due to complications. CONCLUSIONS: The KD was a safe and effective therapy for seizures in children with intractable epilepsy and RC complex defects.     

Myoclonus epilepsy and ragged-red fibers: blood mitochondrial DNA heteroplasmy in affected and asymptomatic members of a family

By a rapid PCR-based method to assess the 8344 mtDNA mutation associated with MERRF disease, we have studied DNA from blood samples of 10 individuals belonging to a family spanning four generations in which one patient showed the complete MERRF phenotype, three other members were less severely affected, while the remaining were unaffected. The percentage of mutant mtDNA was quantified by laser-densitometric scanning of the negative photographic sheets of the agarose gels. The results showed that the MERRF patient had 53% of mutated mtDNA while the two less affected patients had 62% and 14% of mutated mtDNA, respectively. However, a high percentage of mutated genomes (up to 64%) was also found in some unaffected relatives. These results show that although on one hand the mutation is probably the primary cause of the disease, on the other hand the relative amount of mutated mtDNA in blood samples is not indicative of its clinical expression.     

Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells

BACKGROUND: Apoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVE: To measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGN: Randomized controlled animal study. SETTING: Zhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALS: A total of 36 female SD rats of 50 days old and clean grade and weighing (150±10) g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group (n =6) and N-methyl-N-nitrosourea (MNU) group (n =30), and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODS: The experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: Expression of caspase-3 mRNA in retina of rats in the two groups. RESULTS: A total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points (12 hours, 1, 2, 3 and 5 days) after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5th day. CONCLUSION: The expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.     

Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction

We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1374–1381, 1998     

线粒体肌病和脑肌病患者骨骼肌细胞线粒体DNA缺失分析

目的为了检测线粒体肌病和脑肌病患者的骨骼肌细胞的线粒体ＤＮＡ的缺失情况。方法从６例原发性线粒体肌病和１例脑肌病患者的骨骼肌活检标本中，提取总ＤＮＡ，以线粒体ＤＮＡ全长为探针进行分子杂交。结果发现１例ＭＥＲＲＦ患者有５ｋｂ的线粒体ＤＮＡ基因缺失，另１例线粒体肌病患者有１５ｋｂ的线粒体ＤＮＡ基因缺失，剂量分析表明缺失型线粒体ＤＮＡ分别占总线粒体ＤＮＡ的１９．３％和１０．７％。结论线粒体ＤＮＡ基因缺失是线粒体疾病的重要病因之一     

帕金森病患者线粒体功能缺陷的研究

目的　探讨帕金森病 (PD)患者线粒体功能缺陷类型及其基因突变基础。方法　用溴化乙啶抑制人食管癌细胞系的线粒体DNA复制 ,制备出无线粒体DNA细胞系。将 2 0例PD患者组及2 0名正常对照组血小板与之融合 ,用选择性培养液挑选出融合细胞 ,大量培养后用极谱法测定线粒体酶复合体活性及抗氰呼吸。结果　以苹果酸和谷氨酸为底物时患者组 ( 1.2 5± 0 .0 8)明显低于正常对照组 ( 1.75± 0 .0 8) ,降低 2 8.6% ;以琥珀酸、维生素C和TMPD为底物时患者组与正常对照组差别无统计学意义。这些结果表明 ,线粒体酶复合体II～Ⅴ活性正常 ,以苹果酸和谷氨酸为底物的氧耗率的降低来源于酶复合体I活性受损。由于融合细胞核背景一致 ,其线粒体功能仅受mtDNA影响 ,因此本试验发现的患者酶复合体I缺陷来源于mtDNA的突变。抗氰呼吸PD患者组平均为 ( 8.76±0 .2 5 ) % ,正常对照组平均为 ( 6.2 0± 0 .10 ) % ,差异有显著意义 (P <0 .0 5 )。结论　PD患者线粒体酶复合体I活性降低 ,来源于线粒体DNA突变 ,可能导致自由基增多 ,在PD神经元变性中起重要作用。