人脑胶质细胞瘤恶性进展相关基因表达谱中DNA修复基因分析

目的探讨DNA修复基因的变化及其在人脑胶质瘤恶性进展过程中的作用.方法采用cDNA芯片检测同一患者初发和两次复发的胶质瘤组织与正常脑组织中基因表达的差异,建立胶质瘤恶性进展相关基因表达谱.根据GeneBank、GeneCards上检索的资料分析其中DNA修复基因的变化.结果确立与DNA修复相关的基因17条,分别在肿瘤恶性进展的不同阶段上调或下调.结论一些DNA修复基因可能参与了人脑胶质瘤的放疗、化疗耐受及恶性进展过程.     

胶质瘤细胞分化与恶性进展相关分子研究

目的 探讨胶质瘤细胞分化与恶性进展的分子变化,为进一步研究胶质瘤发生的分子病因创造条件.方法 在自建的人脑胶质瘤细胞诱导分化和神经节细胞胶质瘤恶性转化相关基因表达谱中,采用生物信息学聚类方法筛选胶质瘤细胞分化与恶性进展密切相关的新基因,进而分别用反向多点杂交和逆转录聚合酶链反应(RT-PCR)技术在分化程度不同的临床胶质瘤标本中加以验证.又将最感兴趣的基因用脂质体法转染胶质瘤细胞,观察其调控分化效应.结果 随胶质瘤细胞诱导分化而表达量增高的基因有ADP核糖转移酶样蛋白3(ADP-ribosyltransfrase like protein3, Adprt3)、NESH蛋白(new molecule containing SH3 domain)、维甲酸和干扰素诱导的细胞凋亡调控蛋白(genes associated with retinoid IFN induced mortality-19, GRIM-19)、半胱胺天冬酶-1(Caspase-1)和erbB-2转导蛋白-1(transducer-1 of erbB-2, TOB1)共5条;表达量下降的有p8蛋白(p8 protein)、Src样结合蛋白(Src-like adoptor protein, SLAP)、过氧化物酶体增殖受体结合蛋白(peroxisome proliferation-activated receptor binding protein, PPARBP)、尤文肉瘤断列点区-1(Ewing sarcoma breakpoint region-1, EWSR-1)、细胞呼吸因子-1(nuclear repiratory factor-1, NRF-1)、单核细胞超化蛋白-1(monocyte chemotactic protein-1, MCP-1)共6条.随神经节细胞胶质瘤恶化表达量增高的有锌指蛋白157(Zinc finger protein 157)、胆碱转运因子-2(CTL2)、T54蛋白(T54 protein)、血清素(albumin)、肌球蛋白轻链(myosin light polypeptide)、血清蛋白P(serum amy loid P component)、肿瘤坏死因子受体调节因子-1(TNF receptor shedding regulator, ARTS-1)、嗜酸细胞源性神经毒素(eosinophil-derived neurotoxin, EDN)和剪切刺激因子(cleavage stimulation factor, CstF)共9条基因,下降的有胆碱激酶样蛋白(choline kinase-like protein)和DNA多聚酶p12亚基(DNA polymerase epsilon p12 subnit)共2条.结论 我们筛选到的与胶质瘤细胞分化或恶性进展密切相关的新基因共22条,尤其是ARTS-1、TOB1等基因可进一步用于基因转染、RNA干扰和基因敲除等作为调控胶质瘤细胞分化的关键基因进行研究.     

Livin基因短发卡RNA表达质粒的构建及其对胶质瘤Livin基因表达的影响

目的构建凋亡抑制基因livin基因的特异性短发卡RNA（siRNA）真核表达载体,并观察其在人脑胶质瘤细胞中对livin基因表达的抑制。方法设计有小发夹结构的2条livinβ siRNA对应的DNA序列,将其克隆入pSliencer 3.1质粒,构建重组质粒pSliencer-livinβ,对重组质粒进行酶切分析和DNA序列测定。以脂质体法将pSliencer-livinβ转染人胶质瘤细胞。采用RT-PCR和Western-blot检测Livinβ蛋白的表达,筛选最有效的一组pSliencer-livinβ质粒。结果酶切及测序证实质粒pSliencer-livinβ构建成功。转染后胶质瘤细胞livinβmRNA和蛋白表达均受到明显抑制。结论成功构建livinβ基因的特异性短发卡RNA（siRNA）真核表达载体能够显著抑制人胶质瘤细胞livinβ基因的表达。     

胶质瘤恶性进展相关基因PASG敲除后基因表达谱的改变

目的探索胶质瘤恶性进展相关PASG基因与胶质瘤发生发展的关系。方法选择性敲除PASG基因7.126kb基因组DNA序列(含该基因第10～12个外显子),并从形态学及全基因组基因表达水平观察基因敲除后的改变。结果PASG基因敲除鼠全基因组表现为低甲基化水平。海马区皮层的大锥体细胞存在细胞形态、排列层次和数量的异常。鼠脑全基因组差异基因表达谱分析显示:PASG基因敲除后,表达差异超过1.6倍的基因共有123条,其中61条表达下调,62条表达上调。结论PASG基因作为上游调控基因,通过改变基因组甲基化水平调控下游一系列细胞增殖分化相关基因表达。PASG基因表达异常导致下游相关基因的表观遗传修饰改变可能是胶质瘤发生发展的早期分子事件,值得进一步深入研究。     

不同恶性程度胶质瘤细胞基因的差异表达

弄清胶质瘤基因表达谱的变化，对深入研究胶质瘤发生、发展的分子机制有重要意义。本实验用基因芯片技术，比较不同恶性程度的人胶质瘤细胞系CHG-5（WHO分级Ⅱ级）和SHG-44（WHO分级Ⅳ级）的差异表达基因，为胶质瘤分子机制的进一步研究提供基础资料，以期改善胶质瘤的诊断和治疗。     

TaqMan低密度表达芯片检测DNA损伤修复基因在原发胶质瘤中的表达研究

目的运用低密度表达谱芯片检测人脑原发胶质瘤组织中DNA损伤修复基因的表达情况,进一步分析其表达变化的意义。方法使用TaqMan低密度表达芯片技术检测27个DNA损伤修复基因在40例不同级别原发胶质瘤组织和10例正常脑组织中的表达情况,并通过统计学分析其在不同级别胶质瘤和正常脑组织中的表达差异。结果与正常脑组织相比较,有13个DNA损伤修复基因在Ⅱ、Ⅲ、Ⅳ级胶质瘤中均表达下调,包括ERCC1、ERCC2、ERCC3、ERCCA、MGMT、MLH1、MLH3、NTHL1、OGG1、RAD50、SMUG1、XRCC4、XRCC5(P<0.05)。MSH2、MSH6、NUDT1和XRCC3只在Ⅱ级和Ⅲ级胶质瘤中表达下凋;MRE11A和MUS81只在Ⅲ级和Ⅳ级胶质瘤中表达下调。PMS2、RAD52和XRCC1只在Ⅲ级胶质瘤中表达下调,而UNG只在Ⅱ级中表达下调。结论TaqMan低密度表达芯片为多个基因的同时定量表达提供了一种准确、快速、有效的多变量检测技术,可用于发现新的肿瘤相关基因。大量的DNA损伤修复基因的表达下调,与胶质瘤的发生密切相关。     

ATRX基因突变在胶质瘤发生和发展中作用的研究进展

α地中海贫血伴智力低下综合征X连锁(ATRX)基因是位于X染色体的α地中海贫血伴智力低下综合征的致病基因,在染色质重塑、基因组和端粒稳定性的维持中起重要作用。约30%的胶质瘤患者存在ATRX基因突变及其编码的ATRX蛋白缺失,是世界卫生组织(WHO)Ⅱ、Ⅲ级星形细胞瘤以及继发性胶质母细胞瘤的特征性分子改变。此外,ATRX基因突变可作为评估胶质瘤患者预后的指标。ATRX缺失的胶质瘤细胞通过端粒替代延长机制维持端粒的长度,其端粒DNA的损伤重,克服端粒DNA的损伤可能是此类胶质瘤快速恶性进展的重要机制,针对端粒DNA的损伤修复进行干预可能达到靶向治疗的效果。     

胶质细胞分化障碍与胶质瘤发生和发展相关分子研究

我们已经在细胞形态学等方面证明,无论在体外还是动物体内诱导分化剂可使分化差的胶质瘤细胞向分化好的方向转化;而在人体内,分化相对好的神经细胞胶质瘤能自发地向分化很差的多行性胶质母细胞瘤恶性转化,但其发生的分子机制不清。为探讨胶质瘤发生和发展的分子变化,进一步研究胶质瘤发生的分子病因创造条件,在自建的人脑胶质瘤细胞诱导分化和神经节细胞胶质瘤恶性转化相关基因表达谱中,采用生物信息学聚类分析方法筛选胶质瘤分化与去分化密切相关的新基因,进而分别用DotBlot和RT-PCR在分化程度不同的临床胶质瘤标本和处于不同分化阶段体…     

Hepatic Considerations in the Use of Antiepileptic Drugs

Summary: Virtually all of the major antiepileptic drugs (AEDs) can cause hepatotoxicity, although fatal hepatic reactions are rare. The mechanisms, incidences, and risk profiles for such reactions differ from drug to drug. With carbamazepine and phenytoin, hepatotoxicity may be due to drug hypersensitivity. Although the profiles of patients at risk have not been well-defined for these two antiepileptic drugs, it would appear from reports in the literature that older adolescents and adults are at higher risk than children of developing serious or fatal hepatotoxicity. Once hepatotoxicity develops, mortality rates are 10–38% with phenytoin and 25% for carbamazepine. The risk profile for valproate fatal hepatotoxicity has been more clearly defined. Those at primary risk of fatal hepatic dysfunction are children under the age of 2 years who are receiving multiple anticonvulsants and also have significant medical problems in addition to severe epilepsy. The risk is considerably lower for patients over the age of 2 years on valproate monotherapy. In contrast to the risk profile with other AEDs, adults receiving valproate as monotherapy have the lowest risk of hepatotoxicity. Fatal hepatic dysfunction coincident with valproate may be the result of aberrant drug metabolism. Concomitant use of AEDs that induce microsomal P450 enzymes (e.g., phenytoin and phenobarbital) may enhance the production of a toxic metabolite, and hence the greater risk of hepatotoxicity with polypharmacy.     

Vascular Malformations and Epilepsy: Clinical Considerations and Basic Mechanisms

Summary: Vascular malformations (VMs) are associated with epilepsy. The natural history of the various VMs, clinical presentation, and tendency to provoke epilepsy determine treatment strategies. Investigations have probed the mechanisms of epileptogenesis associated with these lesions. Electrophysiologic changes are associated with epileptogenic cortex adjacent to VMs. Putative pathophysiologic mechanisms of epileptogenesis include neuronal cell loss, glial proliferation and abnormal glial physiology, altered neurotransmitter levels, free radical formation, and aberrant second messenger physiology.     

Diagnostic Difficulties and Treatment Implications

Summary: Differentiation between types of epileptic seizures has been aided in recent years by the introduction of intensive neurodiagnostic techniques and the development of increasingly detailed classification systems. Paradoxically, these developments have not simplified the task of matching the appropriate antiepileptic drug to a particular seizure type. It is reasonable to assume that anticonvulsant drugs will have different effects on different types of seizures, but faulty, circular reasoning can enter the picture if one also assumes that responses of seizures to different drugs signify different seizure types. There are several examples of differential diagnoses that can fall prey to this problem, including the diagnosis between partial seizures with secondary generalization and generalized tonic-clonic seizures, and the diagnosis between complex partial seizures and absence seizures with automatisms, among others. Considerations of etiology in future classification systems can further complicate the problem: should one then choose an anticonvulsant drug on the basis of individual seizure type or on the basis of the type of epilepsy? Ramifications of this issue extend even to the drug approval process. Official sanction is not given for use of a drug for a seizure type not included in the original efficacy studies, even if later scientific evidence shows that seizure type to be related to a type that is included. New trials must be undertaken. These problems arise from how we choose to classify seizures.     

Cognitive Dysfunction Associated with Antiepileptic Drug Therapy

Summary: Epilepsy is frequently associated with cognitive dysfunction. However, the reasons for this correlation are unclear. Possible influential factors include patient age; duration, frequency, etiology, and type of seizures; hereditary factors; psychosocial issues; and antiepileptic drug (AED) therapy. Whereas many of these factors are beyond the physician's control, AED therapy is one element that can be addressed in treatment decisions by recognizing the potential cognitive effects of particular AEDs. For example, phenobarbital impairs memory and concentration; phenytoin affects attention, problem solving ability, and performance of visuomotor tasks. In contrast, carbamazepine may affect concentration, while valproate would appear to have minimal effects on cognition. Moreover, cognitive effects of AEDs are amplified with coadministration of multiple anticonvulsants (polytherapy). A review of studies on the cognitive effects of monotherapy with AEDs, as opposed to those of polytherapy, provides evidence that drug-related cognitive dysfunction can be reversed if patients are switched to a simpler therapeutic regimen. Future research should be directed toward developing reliable measures for assessing and monitoring cognition, and understanding the particular cognitive side effects of each AED. Physicians also need to revise their opinions about which side effects are "tolerable" for epileptic patients.     

Epilepsy and anomalies of neuronal migration: MRI and clinical aspects

Neuronal migration disorders are the result of disturbed brain development. In such disorders, neurons are abnormally located. In diagnosing these conditions, magnetic resonance imaging is superior to any other imaging technique. This enables us to improve our knowledge of the clinical correlates of neuronal migration. With reference to migrational disorder, a retrospective study of all 303 patients with epileptic seizures referred for magnetic resonance imaging during a 3-year period was performed, 13 patients (aged 12-41, mean age 27) were identified. They represent 4.3% of the entire study group. Of the patients with known epilepsy, 6.7% and of the mentally retarded, 13.7% had migrational disorders. Four patients had schizencephaly as the dominant finding, one was classified as hemimegalencephaly, 2 had isolated heterotopias, and 6 had localized pachy- and/or poly-microgyria. The clinical pictures are complex. Ectopias of grey matter are recognised foci of epilepsy, but from an epileptological and a clinical viewpoint little attention has been given to these disorders. The present study shows that malmigration is not rare in epilepsy patients, especially not in the mentally retarded.     

Carbamazepine Efficacy and Utilization in Children

Summary: Carbamazepine is effective for preventing partial and generalized tonic-clonic seizures in children. Although absence epilepsies are more common in children than adults, an estimated 80% of children with epilepsy have seizure types or epilepsies that are potentially responsive to carbamazepine. The differential diagnosis of ictal staring is an especially important issue in children because absence and atypical absence seizures are more prevalent in children than adults. Age-related pharmacokinetic differences and drug interactions are major considerations in children. On average, children have higher clearance rates of carbamazepine, shorter half-lives, and higher ratios of carbamazepine-10, 11-epoxide to carbamazepine than adults. In addition, children with severe epilepsy are more likely to require multiple-drug therapy, which can lead to complex drug interactions. When carbamazepine is administered along with valproate, drug protein binding interactions can cause intermittent side effects.     

Carbamazepine Efficacy in Adults With Partial and Generalized Tonic-Clonic Seizures

Summary: Carbamazepine and phenytoin are drugs of choice in initial monotherapy for adult partial and secondarily generalized tonic-clonic seizures. These designations reflect the results of the Veterans Administration Epilepsy Cooperative Study Group of 1985. An earlier comparative study of carbamazepine and phenytoin by Ramsay and associates found both drugs equally effective in controlling new-onset seizures. Among the advantages of carbamazepine is that it causes relatively few cognitive and dysmorphic side effects. Its disadvantages are its unavailability in parenteral formulation and its metabolic autoinduction. The latter must be compensated for by planned dosage increases to maintain therapeutic plasma steady-state levels during the first 2 or 3 months of treatment. Carbamazepine is judged a drug of choice in the treatment of these secondarily generalized tonic-clonic seizures, and the drug of choice in children, adolescents, and women susceptible to the dysmorphic side effects associated with other anticonvulsant agents.     

Etiologic and Preventive Aspects of Epilepsy in the Child–Bridging the Gap Between Laboratory and Clinic

Summary: Four broad categories of basic phenomena are pertinent to developing ways to prevent epilepsy. These include mechanisms of epileptogenesis, ictal initiation and temporary entrainment by the seizure discharge of normally functioning brain, seizure propagation, and control mechanisms that function both to restrain the cascade of epileptic events culminating in a seizure and to arrest the epileptic event and restore the interictal state. In newborns and children, hypoxia-ischemia is a major factor leading to epileptogenesis, and several schemes are proposed to classify, quantify, and prevent hypoxic-ischemic encephalopathy. Control mechanisms must be better understood in order to develop prophylactic recommendations for epilepsy, and an experimental model of "kindling antagonism" may increase our understanding of these. Programs of prevention of seizures in children will evolve only if basic researchers and clinicians work productively together to develop an adequate understanding of factors important in epileptogenesis and antiepileptogenic control mechanisms.     

Predisposing and Causative Factors in Childhood Epilepsy

Summary: We review information from large studies of defined populations, examining the role of known factors and especially of prenatal and perinatal factors in contributing to nonfebrile seizure disorders of early childhood. We depend especially, but not exclusively, on the recently completed analyses from the Collaborative Perinatal Project of the National Institute of Neurological and Communicative Disorders and Stroke, the NCPP. About 4% of children in the NCPP who had at least one non-febrile nonsymptomatic seizure by the age of 7 years had a previous seizure during acute neurologic illness, such as meningitis or during the acute illness after trauma. Many such seizures should potentially be preventable. Of children with seizures, 10% had had a neonatal seizure and 13% had had a febrile seizure. Among the hundreds of prenatal and perinatal factors explored as predictors of childhood seizure disorders, the principal predictors identified were congenital malformations of the fetus, cerebral and noncerebral; family history of certain neurologic disorders; and neonatal seizures. In agreement with the British National Child Development Study, labor and delivery factors in the NCPP appeared to contribute very little to childhood seizure disorders. Maldevelopment, rather than damage at birth to an initially intact nervous system, appeared to be the more common mechanism. Most seizure disorders of early childhood remained unexplained by the large set of prenatal and perinatal characteristics examined.     

Classification of methods in transcranial Electrical Stimulation (tES) and evolving strategy from historical approaches to contemporary innovations

Transcranial Electrical Stimulation (tES) encompasses all methods of non-invasive current application to the brain used in research and clinical practice. We present the first comprehensive and technical review, explaining the evolution of tES in both terminology and dosage over the past 100 years of research to present day. Current transcranial Pulsed Current Stimulation (tPCS) approaches such as Cranial Electrotherapy Stimulation (CES) descended from Electrosleep (ES) through Cranial Electro-stimulation Therapy (CET), Transcerebral Electrotherapy (TCET), and NeuroElectric Therapy (NET) while others like Transcutaneous Cranial Electrical Stimulation (TCES) descended from Electroanesthesia (EA) through Limoge, and Interferential Stimulation. Prior to a contemporary resurgence in interest, variations of transcranial Direct Current Stimulation were explored intermittently, including Polarizing current, Galvanic Vestibular Stimulation (GVS), and Transcranial Micropolarization. The development of these approaches alongside Electroconvulsive Therapy (ECT) and pharmacological developments are considered. Both the roots and unique features of contemporary approaches such as transcranial Alternating Current Stimulation (tACS) and transcranial Random Noise Stimulation (tRNS) are discussed. Trends and incremental developments in electrode montage and waveform spanning decades are presented leading to the present day. Commercial devices, seminal conferences, and regulatory decisions are noted. We conclude with six rules on how increasing medical and technological sophistication may now be leveraged for broader success and adoption of tES.