Rac1在胶质瘤中的表达及其与胶质瘤细胞迁移、侵袭的关系

目的观察Rac1在胶质瘤组织中的表达,探讨其在胶质瘤迁移、侵袭中的作用。方法运用Western blot、RT-q PCR检测65例胶质瘤脑组织和8例正常脑组织中Rac1蛋白和m RNA表达。体外培养人胶质瘤细胞系U251,将细胞分为三组(空白对照组、Rac1抑制剂组、Rac1类似物组)。通过细胞划痕实验和Transwell细胞侵袭实验检测三组细胞运动能力的变化。结果Rac1蛋白和m RNA在正常脑组织中弱或无表达,在胶质瘤组织中高表达,且分别与病理级别成正相关(r=0.956,r=0.857)。三组细胞中,Rac1类似物组的运动能力最强,Rac1抑制剂组最弱(P0.05)。结论 Rac1在胶质瘤组织中的高表达与胶质瘤的迁移、侵袭密切相关,可作为一个反映胶质瘤运动能力和恶性程度的指标。     

胶质瘤干细胞外泌体通过PI3K/Akt信号通路促进胶质瘤U87细胞侵袭、迁移

目的 探讨胶质瘤干细胞（GSCs）外泌体（exo）对胶质瘤U87细胞侵袭、迁移的影响。方法 取对数生长期U87细胞,应用干细胞培养液分离、培养胶质瘤U87细胞中GSCs并提取、鉴定GSCs-exo。取对数生长期U87细胞随机分为4组:对照组和高、中、低GSCs-exo浓度组（分别加入20、40、80 μg/ml的GSCs-exo）。Transwell实验检测U87细胞侵袭能力,划痕实验检测U87细胞迁移能力,免疫印记法检测U87细胞磷脂酰肌醇-3激酶（PI3K）/蛋白激酶B（Akt）、L1CAM的表达水平。结果 成功分离GSCs并获得GSCs-exo。GSCs-exo组U87侵袭细胞数、细胞迁移率明显高于对照组（P<0.05）,L1CAM、PI3K及p-AKt/Akt表达水平明显高于对照组（P<0.05）,而且均呈浓度依赖性（P<0.05）。结论 GSCs-exo可促进胶质瘤侵袭与转移,其机制可能与上调L1CAM、PI3K表达,促进Akt磷酸化有关。     

内皮抑素和血管生成抑素融合基因修饰的单纯疱疹病毒对胶质瘤干细胞的体外实验研究

目的 探讨内皮抑素和血管生成抑素( Endo - Angio)融合基因修饰的单纯疱疹病毒(VAE)对胶质瘤干细胞(GSCs)的体外溶瘤作用。方法 新鲜高级别（WHOⅢ级、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs,采用流式细胞术检测其中CD133阳性细胞数,单克隆形成实验检测GSCs的自我更新能力,免疫荧光技术检测未分化GSCs的Nestin及分化后GFAP、NF和MAG的表达;MTS法检测VAE对GSCs增殖活性的影响,RT - PCR和Western blot检测Endo - Angio融合蛋白的表达并检测其对人脑微血管内皮细胞(HBMEC)增殖的影响;最后观察病毒处理后仍存活细胞的自我更新和诱导贴壁情况。结果 (1)从20例高级别胶质瘤标本中培养出4例GSCs,能够表达CD133及Nestin,具有自我更新能力和多向分化能力。(2)VAE能够感染GSCs,4例GSCs增殖活性明显下降(P＜0.05)。(3)VAE感染GSCs 48 h后,基因水平及蛋白水平都有Endo- Angio融合蛋白表达,该融合蛋白使HBMEC增殖活性明显下降(P＜0.05)。(4)VAE感染后仍存活的细胞不再具有形成GSCs球的能力,加入血清诱导后也不再贴壁生长。结论 在体外,VAE能够显著抑制GSCs活性,能够表达具有生物活性的Endo-Angio融合蛋白,为脑胶质瘤溶瘤病毒治疗开辟了新的途径。     

阿托伐他汀减少小胶质细胞MMP-2诱导的胶质瘤侵袭和迁移

目的 研究阿托伐他汀对小胶质细胞促胶质瘤侵袭和迁移作用的影响及对胶质瘤侵袭和迁移的抑制作用.方法 CCK-8法检测梯度浓度阿托伐他汀对原代人小胶质细胞和U87胶质瘤细胞活性影响;胶质瘤条件培养基(GCM)激活小胶质细胞制备小胶质细胞条件培养基(MCM),Transwell细胞迁移实验和肿瘤侵袭实验检测MCM干预下U87侵袭和迁移能力,同时使用阿托伐他汀干预小胶质细胞激活过程,检测U87侵袭和迁移能力改变;明胶酶谱法检测阿托伐他汀干预下U87MMP-2和MMP-9表达,及阿托伐他汀与GCM联合干预下小胶质细胞MMP-2和MMP-9表达.结果 10-5mol/L阿托伐他汀对U87和小胶质细胞活性无明显影响(P＞0.05).U87在MCM中孵化24h,迁移和侵袭细胞数均较对照组增加(P＜0.001).U87在阿托伐他汀MCM中孵化24h,迁移和侵袭细胞数均较MCM组显著减少(P＜0.001).U87在阿托伐他汀中孵化24h,迁移和侵袭细胞数略微低于对照组(P＜0.05).明胶酶谱提示阿托伐他汀干预24h,U87和GCM激活的小胶质细胞MMP-2水平降低(P＜0.001).结论 阿托伐他汀对小胶质细胞MMP-2诱导的胶质瘤侵袭和迁移有强烈抑制作用,同时也能抑制胶质瘤本身的侵袭和迁移.     

胶质瘤干细胞来源的外泌体对血管内皮细胞增殖和迁移的影响

目的探讨胶质瘤干细胞(glioma stem cells,GSCs)来源的外泌体对人脑微血管内皮细胞(HBMECs)的增殖和迁移的影响。方法培养和分离人胶质瘤U87细胞系来源的胶质瘤干细胞(GSCs),对培养的肿瘤球细胞及其诱导分化的细胞采用免疫组织化学染色的方法鉴定。然后提取胶质瘤干细胞分泌的外泌体(GSCs-exo),分别添加不同浓度的GSCsexo (20μg/ml、40μg/ml、60μg/ml)处理人脑微血管内皮细胞(HBMECs)。在处理结束后,采用CCK-8法检测GSCs-exo对血管内皮细胞增殖的影响、Transwell小室检测GSCs-exo对细胞迁移能力的影响。结果肿瘤球细胞培养状态下呈悬浮生长,免疫组织化学荧光染色法证明培养的肿瘤球细胞中特异性表达CD133和Nestin,其诱导分化的细胞染色可见GFAP和Neun表达阳性。随着GSCs-exo浓度的升高,促血管内皮细胞的增殖能力逐渐增强,迁移能力也大大提高(P 0. 05)。结论在胶质瘤微环境中,胶质瘤干细胞来源的外泌体可以促进血管内皮细胞的增殖和迁移,GSCs-exo可能是胶质瘤血管形成的关键因素。     

人脑胶质瘤干细胞分化为血管内皮细胞的实验研究

目的 探讨体内外原代培养的人脑胶质瘤干细胞(GSCs)与胶质瘤新生血管内皮细胞的关系.方法 新鲜高级别(WHOⅢ级、Ⅳ级)的人脑胶质瘤标本经原代培养获取GSCs,免疫组化法检测其肿瘤干细胞及干细胞标记物Nestin的表达;鉴定后的GSCs经低氧诱导,免疫荧光法检测其诱导分化后内皮细胞标记物CD31、CD144和胶质瘤细胞标记物GFAP的表达,RT-PCR和Western-blot法检测其CD31的表达;建立胶质瘤干细胞皮下荷瘤裸鼠模型,免疫组化技术检测模型中人来源的CD31的表达.结果 (1)悬浮生长的胶质瘤干细胞球样细胞经免疫组化鉴定Nestin表达阳性;(2)低氧诱导后的GSCs能够表达CD31、CD144,有些细胞能够同时表达CD144和GFAP;(3)RT-PCR检测发现GSCs在诱导前后都有CD31 mRNA的表达,而Western-blot检测到只有诱导后的GSCs有CD31蛋白的表达;(4)胶质瘤干细胞荷瘤裸鼠模型的肿瘤组织中部分微血管抗人CD31抗体染色阳性.结论 胶质瘤干细胞不仅在体外低氧条件下可分化为内皮细胞,在体内微环境条件下同样可分化为血管内皮细胞,并参与胶质瘤新生组织的血液供应.     

槐定碱通过Wnt/β-catenin信号通路抑制人胶质瘤U87细胞迁移、侵袭

目的 探讨槐定碱对人胶质瘤U87细胞迁移、侵袭能力的影响及其作用机制。方法 体外培养人胶质瘤U87细胞,加入槐定碱[0 mg/ml（对照组）,1.0 mg/ml,2.0 mg/ml,3.0 mg/ml]共培养24 h,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,免疫印迹法检测细胞β-catenin、E-cadherin、Vimentin和MMP-9蛋白表达,RT-PCR检测细胞Vimentin、MMP-9 mRNA表达。结果 槐定碱明显抑制U87细胞迁移、侵袭能力（P<0.001）,而且随浓度增加抑制作用明显增强（P<0.001）。槐定碱明显抑制U87细胞β-catenin蛋白、Vimentin和MMP-9 mRNA和蛋白表达（P<0.01）,明显增强E-cadherin蛋白表达（P<0.01）,而且随浓度增加抑制作用明显增强（P<0.01）。结论 槐定碱能抑制人胶质瘤U87细胞的迁移和侵袭,机制可能与抑制Wnt/β-catenin信号通路的活性,阻断细胞上皮间质转化过程。     

siRNA下调Caveolin-1表达对体外培养C6胶质瘤细胞影响

目的 观察siRNA下调Caveolin-1(Car1)表达的效果及对体外培养C6胶质瘤细胞侵袭和迁移能力的影响.方法 构建Car1 mRNA质粒,转染体外培养c6胶质瘤细胞,利用Transwell体外侵袭实验和"划痕"实验分别检测细胞的侵袭和迁移能力,采用Western blot和RT-PCR的方法检测Cav1、EGFR、Pyk2的表达情况.结果 所构建的Cav1 siRNA质粒转染体外培养C6胶质瘤细胞可明显下调Cav1的表达,干扰成功后C6胶质瘤细胞的侵袭能力和迁移能力明显下降,同时EGFR和Pyk2的表达明显减少.结论 siRNA下调Cav1可降低体外培养C6胶质瘤细胞的侵袭和迁移能力.     

组蛋白甲基转移酶对人脑胶质瘤细胞迁移和侵袭的影响及其机制的研究

目的探讨组蛋白甲基转移酶(SMYD2)在人脑胶质瘤及正常脑组织中的表达及其对人脑胶质瘤细胞U87、U251迁移和侵袭的影响。方法运用免疫组织化学方法检测50例脑胶质瘤及10例正常脑组织中SMYD2蛋白的表达水平。应用小干扰RNA(siRNA)技术分别转染人脑胶质瘤U87、U251细胞,Western blot检测SMYD2蛋白及基质金属蛋白酶-9(MMP-9)、p-p65、TIMP1的表达水平;应用细胞划痕实验及Transwell迁移、侵袭实验检测细胞的迁移和侵袭能力。结果免疫组织化学显示SMYD2在正常组、低级别胶质瘤(Ⅰ~Ⅱ级)组和高级别胶质瘤(Ⅲ~Ⅳ级)组中表达水平的差异有统计学意义(P0.05),且SMYD2蛋白的表达量与肿瘤的恶性程度呈正相关(r=0.635,P0.05)。与Control-siRNA组比较,SMYD2-siRNA组SMYD2蛋白表达量显著降低,MMP9、p-p65显著降低,TIPM1明显增加,差异均有统计学意义(P0.05~0.01)。细胞划痕实验显示,SMYD2-siRNA组较Control-siRNA组细胞的迁移能力显著降低(均P0.01)。Transwell迁移、侵袭实验显示干扰SMYD2后,人脑胶质瘤U87、U251细胞的迁移、侵袭能力显著下降(均P0.05)。结论 SMYD2在胶质瘤中表达,并且其表达水平与胶质瘤恶性程度呈正相关;SMYD2-siRNA靶向干扰了SMYD2的表达,下调SMYD2的表达水平能够降低人脑胶质瘤细胞U87、U251迁移、侵袭的能力。     

钙蛋白酶-1在胶质瘤组织中的表达及其对U87细胞侵袭和迁移的影响

目的 探讨钙蛋白酶-1在人脑胶质瘤组织中的表达及其对U87胶质瘤细胞侵袭和迁移的影响。方法 收集2015年1月至2018年3月手术切除的胶质瘤组织和瘤旁正常脑组织各57例。体外培养U87细胞,将阴性对照小干扰RNA（NC组）和钙蛋白酶-1小干扰RNA（钙蛋白酶-1组）转染至人胶质瘤细胞株U87,以未转染的U87细胞为对照组。采用RT-PCR和免疫印迹法检测mRNA和蛋白表达水平,Transwell实验检测U87细胞侵袭能力,划痕实验评估U87细胞迁移能力。结果 胶质瘤组织钙蛋白酶-1 mRNA和蛋白表达水平均显著高于瘤旁正常脑组织（P<0.05）。钙蛋白酶-1组U87细胞钙蛋白酶-1 mRNA和蛋白表达水平、细胞侵袭数目、细胞迁移率以及转化生长因子-β、基质金属蛋白酶（MMP）-2和MMP-9 mRNA表达水平均明显低于对照组和NC组（P<0.05）,而对照组和NC组均无统计学差异（P>0.05）。结论 钙蛋白酶-1在人脑胶质瘤中表达升高,而抑制钙蛋白酶-1表达可能通过影响上皮间质转化,抑制胶质瘤细胞侵袭和迁移。     

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.     

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.     

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.     

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.     

Neonatal Seizures: Problems in Diagnosis and Classification

Summary: The clinical identification of neonatal seizures is critical for the recognition of brain dysfunction; however, diagnosis is often difficult because of the poorly organized and varied nature of these behaviors. Current classification systems are limited in their ability to communicate motor, autonomic, and electroencephalo-graphic features of seizures precisely and to provide a basis for uniform effective diagnosis, therapy, and determination of prognosis. Recent investigations of neonates, utilizing bedside electroencephalographic/polygraphic/ video monitoring techniques, have provided the basis for improved diagnosis and classification of seizures in the newborn. These studies have demonstrated that not all clinical phenomena currently considered to be seizures require electrocortical epileptiform activity for their initiation or elaboration. In addition, the specific clinical character of the phenomena considered to be seizures, the clinical state of the infant, and the character of the EEG indicate the probable pathophysiological mechanisms involved and suggest probable etiologies, prognosis, and therapy. Similarities between animal models that demonstrate reflex physiology and neonates with motor automatisms and tonic posturing suggest that these clinical behaviors may not be epileptic in origin but, rather, primitive movements of progression and posture mediated by brainstem mechanisms. Although not all clinical behaviors currently considered to be neonatal seizures may have similar pathophysiological mechanisms, they are clinically significant because they all indicate brain dysfunction.     

Valproate Monotherapy in the Management of Generalized and Partial Seizures

Summary: For decades, therapeutic tradition has promoted the concept of polypharmacy in the management of epilepsy. In recent years, however, studies have shown that, for most patients, monotherapy can provide comparable or better seizure control than administration of multiple anticonvulsants, while diminishing the potential for adverse reactions, drug interactions, and poor compliance. Valproate is an important monotherapeutic agent that is highly effective in the control of idiopathic primary and secondarily generalized epilepsies, and partial seizures that do not generalize. Comparative studies have found that valproate is at least as effective as phenytoin and carbamazepine in the treatment of generalized and partial seizures. Given the similar efficacy, other factors such as pharmacokinetics and side effects may therefore determine anticonvulsant selection for monotherapy.     

Un nouveau cadre conceptuel de travail pour la psychiatrie

In an attempt to place psychiatric thinking and the training of future psychiatrists more centrally into the context of modern biology, the author outlines the beginnings of a new intellectual framework for psychiatry that derives from current biological thinking about the relationship of mind to brain. The purpose of this framework is twofold. First, it is designed to emphasize that the professional requirements for future psychiatrists will demand a greater knowledge of the structure and functioning of the brain than is currently available in most training programs. Second, it is designed to illustrate that the unique domain which psychiatry occupies within academic medicine, the analysis of the interaction between social and biological determinants of behavior, can best be studied by also having a full understanding of the biological components of behavior.