复方中草药茯苓、白术和当归促进人许旺细胞的增殖

摘要： 背景和目的： 周围神经损伤修复的最佳方式是自体神经移植,然而自体神经移植造成患者的二次损伤使其在临床的应用中受到局限。雪旺细胞是周围神经组织工程研究的重要的种子细胞,需要对雪旺细胞进行体外培养、增殖和传代。由于雪旺细胞的增殖相当缓慢,培养周期长等方面限制。许多研究者转向中医药干预的方法进行探寻。令人欣慰的是：相关的实验传统复方制剂FBD能维持神经元生存和生长并促进BDNF等分泌物。本项研究将依据独特的实验模型,采用研究者选用中国药品生物制品检定所提纯的人参皂甙Rb1,加入体外培养的雪旺细胞中进行实验,观察细胞加药后的生长情况。 方法：利用SD雄性大鼠雪旺细胞株（ISC）,加入不同浓度的FBD,利用MTT比色分析法、RT-PCR, ELISA,测定法检测不同浓度FBD在不同培养时间对体外培养大鼠雪旺细胞增殖的影响。 结果：FBD在20微克/毫升的浓度对雪旺细胞增殖有明显促进作用。而50微克/毫升FBD对细胞增殖的促进作用与对照组相近。 结论 ： FBD有促进体外培养雪旺细胞快速增殖的作用,从而为促进神经损伤的再生途径提供一些新的药物使用基础研究。     

人参皂甙Rb1对体外培养雪旺细胞作用的实验研究

目的研究人参皂甙Rb1对体外培养雪旺细胞增殖分化的影响,探讨其促进神经再生的作用和机制。方法取8个月月龄的新西兰兔的坐骨神经雪旺细胞体外培养第2代,加入不同浓度的人参皂甙Rb1,继续培养10d,显微镜观察计数,绘制各自的增殖曲线。在36h和72h对各组细胞进行流式细胞分析。结果活细胞计数显示:含20μg/ml人参皂甙Rb1组的倍增时间为5.3d,明显优于对照组(P<0.01)。用含20μg/ml人参皂甙Rb1培养36h、72h后雪旺细胞增殖的流式细胞分析,处于S期的雪旺细胞较对照组明显增高(P<0.01)。结论人参皂甙Rb1具有促进体外培养雪旺细胞快速增殖分化的作用,有助于损伤神经的再生。     

人参皂甙Rb1对体外培养雪旺细胞作用的实验研究

目的 研究人参皂甙Rb1对体外培养雪旺细胞增殖分化的影响,探讨其促进神经再生的作用和机制.方法 取8个月月龄的新西兰兔的坐骨神经雪旺细胞体外培养第2代,加入不同浓度的人参皂甙Rb1,继续培养10d,相差显微镜观察计数,绘制各自的增殖曲线.在36h和72h对各组细胞进行流式细胞分析.结果活细胞计数显示：含20μg/ml人参皂甙Rb1组的倍增时间为5.3d,明显优于对照组（P＜0.01）.用含20μg/ml人参皂甙Rb1培养36h、72h后雪旺细胞增殖的流式细胞分析,处于S期的雪旺细胞较对照组明显增高（P＜0.01）.结论 人参皂甙Rb1具有促进体外培养雪旺细胞快速增殖分化的作用,有助于损伤神经的再生.     

吡咯喹啉醌对许旺细胞增殖及Sox10表达的影响

背景：雪旺细胞是神经组织工程的种子细胞,但其体外增殖缓慢,难以满足科研与临床对其的需要,吡咯喹啉醌可以对多种细胞的增值产生促进作用,本课题研究吡咯喹啉醌对雪旺细胞增殖及Sox10表达的影响。 目的：研究吡咯喹啉醌对雪旺细胞的增殖作用并探讨其对雪旺细胞Sox10基因表达的影响。 设计、时间及地点：细胞增殖及基因表达检测,于2009-01/04在武汉大学人民医院中心实验室完成。 材料：出生3～4天SD大鼠20只,由武汉大学医学部实验动物中心提供,DMEM/F12培养基、胎牛血清由Gibco公司（USA）提供,Trizol、PCR引物由Invitrogen公司提供,RT-PCR试剂盒由Fermentas公司提供。 方法 雪旺细胞体外培养及纯化,S-100免疫荧光鉴定雪旺细胞;细胞经无血清培养12h后,加入10nM吡咯喹啉醌继续培养24h观察其形态学改变;加入不同浓度（0,1,10,100,1000,10000nM）吡咯喹啉醌于雪旺细胞培养24h,利用RT-PCR技术检测Sox10的mRNA表达。 主要观察指标：观察雪旺细胞形态学,并对雪旺细胞进行计数,应用RT-PCR方法检测Sox10的表达。 结果 吡咯喹啉醌促使雪旺细胞发生形态学改变,多数呈束状或并排生长,且细胞数目增多;1～1000nM吡咯喹啉醌可使雪旺细胞Sox10表达增高,100nM时表达最高;10000nM时对Sox10的表达表现为抑制作用（P<0.05）。 结论 吡咯喹啉醌可促进雪旺细胞增值并促使其发生形态学改变,且Sox10在PQQ促雪旺细胞增殖过程中表达上调。     

甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响*☆

背景：许旺细胞分泌多种神经营养因子在神经再生中发挥关键作用，但其分泌能力受诸多因素影响，寻找可行方法促进许旺细胞分泌神经生长因子是神经缺损后再生的重要环节。 目的：观察不同浓度甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响。 方法：酶消化法分离培养SD乳鼠许旺细胞，倒置相差显微镜下观察细胞生长情况；传代后，一部分进行S-100蛋白免疫鉴定许旺细胞的纯度；另一部分用细胞计数板调整细胞浓度为1×109 L-1，移入到6孔平底培养板(接种15个孔)继续培养。培养板中培养4 d后4个孔分别加入不同浓度的甲泼尼龙琥珀酸钠(10-3，10-4，10-6，10-8 mol/L)，并设立1个孔为不加药物的空白对照组，分别作用于细胞24，48，72 h后行RT-PCR检测，观察许旺细胞24，48，72 h神经生长因子mRNA水平的变化。 结果与结论：原代细胞培养至第7天数量明显增加，细胞铺满培养瓶底部80%以上；传代细胞形态为梭形，有2个细长的突起，荧光染色阳性，成纤维细胞为圆形或扁圆形，荧光染色阴性。RT-PCR检测结果显示，10-8 mol/L的甲基强地松龙作用72 h分泌的神经生长因子与空白对照组及其他浓度、时间组相比均表达增加(P < 0.05)；10-3 mol/L甲基强地松龙在各时间段分泌的神经生长因子与空白对照组及其他浓度、时间组相比表达均减少(P < 0.05)。提示高浓度的甲泼尼龙琥珀酸钠抑制许旺细胞分泌神经生长因子，长时间、低浓度的甲泼尼龙琥珀酸钠则促进许旺细胞分泌神经生长因子。     

大鼠雪旺氏细胞支持人胚胎神经干细胞的生长并诱导其分化

目的 探讨大鼠雪旺氏细胞对人胚胎神经干细胞生长和分化的作用。方法 实验分为三组：组一：干细胞生长于培养雪旺氏细胞1天后的培养液中；组二：干细胞与雪旺氏细胞共培养；组三：干细胞生长于DMEM／F12培养液中，观察干细胞的形态学变化和免疫荧光的.组一的神经球分化生长，绝大多数细胞tubulin-β阳性，少数为GalC或GFAP阳性；组二中，在雪旺氏细胞生长密集和稀少的地方，干细胞分别表明为不分化和明显分化两种状态；组三的神经干细胞无法正常生长。结论 大鼠雪旺氏细胞分泌物支持人胚胎神经干细胞的生长并诱导其分化。     

钙通道阻滞剂对体外培养神经瘢痕成纤维细胞增殖及胶原分泌的影响☆

目的：钙通道阻滞剂可抑制瘢痕成纤维细胞的增殖及胶原蛋白的分泌，减少瘢痕的增生。实验拟进一步观察钙通道阻滞剂对体外培养神经瘢痕成纤维细胞增殖及胶原分泌的影响。 方法：实验于2006-03/06在吉林大学中日联谊医院中心实验室完成。①实验材料：Wistar大鼠10只；盐酸维拉帕米注射液为上海禾丰制药有限公司产品。②实验过程及分组：制作大鼠双侧坐骨神经损伤模型，术后2周取损伤处神经瘢痕，按组织块法培养神经瘢痕成纤维细胞，实验所用细胞为4~8代，分4组，其中3组培养基中维拉帕米药物浓度分别为10，50及100 μmol/L，1组为空白对照组。③实验评估：分别用四甲基偶氮唑盐法观察细胞增殖能力、羟脯氨酸比色法测定细胞上清液中的胶原含量和胞浆中的胶原含量。 结果：①四甲基偶氮唑盐法观察细胞增殖结果：钙通道阻滞剂对神经瘢痕成纤维细胞的生长增殖具有明显抑制作用，且呈现剂量依赖性(P < 0.05)。②羟脯氨酸比色法测定胶原含量：钙通道阻滞剂可使分泌到细胞培养上清中的胶原含量明显减少，以100 μmol/L浓度组作用最为显著(P < 0.05);胞浆中的胶原含量增加，具有明显剂量依赖性，以100μmol/L浓度组作用最为显著（P < 0.05）。 结论：钙通道阻滞剂可以抑制神经瘢痕成纤维细胞的增殖及胶原分泌。     

成骨生长肽对人牙周膜细胞增殖和碱性磷酸酶活性的影响

背景：成骨生长肽是新近发现的一种生长因子,具有促进有丝分裂、促进成骨等作用,在组织损伤后的修复过程中发挥了重要的生理作用。 目的：观察成骨生长肽对体外培养的人牙周膜细胞增殖及其碱性磷酸酶活性的影响。 设计、时间及地点：细胞水平的对比观察实验,于2006-02/06在教育部口腔生物医学工程重点实验室完成。 材料：临床上因正畸需要拔除的牙周健康、无龋的新鲜第1前磨牙牙周膜组织。成骨生长肽购于美国Sigma公司。 方法：在体外培养的人牙周膜细胞中加入10-11,10-10,10-9,10-8 及10-7 mol/L不同浓度的成骨生长肽,用四甲基偶氮唑盐比色法检测细胞的增殖率;以流式细胞仪法检测细胞的增殖周期;应用酶联免疫法检测细胞内碱性磷酸酶活性。 主要观察指标：细胞培养第1,2,3天观察细胞增殖情况;第6天观察细胞内碱性磷酸酶活性。 结果：成骨生长肽浓度在10-11~10-7 mol/L时,对人牙周膜细胞的增殖率有明显的促进作用(P < 0.05),最佳作用浓度为 10-9 mol/L。当成骨生长肽浓度在10-9 mol/L时,能明显提高细胞S期所占的比例,从而加速细胞的增殖活动。成骨生长肽在10-11~10-7 mol/L时,能显著提高细胞内碱性磷酸酶活性(P < 0.05),最佳作用浓度为10-9 mol/L。 结论：成骨生长肽可促进人牙周膜细胞的增殖活性,同时可以提高细胞碱性磷酸酶的活性。     

法舒地尔和RhoA沉默对神经干细胞增殖的影响

背景：Rho及其相关分子在神经轴突生长、分化、延伸及突触形成中起重要作用,阻断和抑制RhoA/ROCK通路可促进神经干细胞的增殖与生长。 目的：观察Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默对大鼠神经干细胞增殖的影响。 方法：体外培养Wistar胎鼠神经干细胞,分6组干预：空白对照组,5,10,15,20 μmol/L 法舒地尔组,siRNA 沉默RhoA基因组。干预后第3天,采用RT-PCR,Western blot检测各组神经干细胞RhoA基因及蛋白的表达。应用MTT比色法观察神经干细胞增殖情况;采用流式细胞术测定神经干细胞周期分布的变化。 结果与结论：15,20 μmol/L 法舒地尔组、siRNA 沉默RhoA基因组神经干细胞RhoA基因及蛋白表达量较5,10 μmol/L 法舒地尔组、空白对照组明显降低(P < 0.05),细胞的生长速度较5,10 μmol/L 法舒地尔组、空白对照组明显增快(P < 0.05),细胞周期G0/G1期减少(P < 0.05),S期细胞数增多(P < 0.05)。当法舒地尔浓度增加到20 μmol/L时对细胞的作用并非随浓度的增加而增强,与15 μmol/L组的差异无显著性意义(P > 0.05)。15,20 μmol/L 法舒地尔组与siRNA 沉默RhoA基因组相比差异无显著性意义(P > 0.05)。说明Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默在体外均能促进神经干细胞增殖,法舒地尔最佳作用浓度为15 μmol/L。     

抑制P-糖蛋白可加强FK506对缺血性脑损伤的保护作用

目的 探讨多药耐药蛋白(P糖蛋白,P-glycoprotein,P-gp)对FK506透过血脑屏障进入脑内浓度及其脑保护作用的影响.方法 用腔内线栓法制造小鼠脑缺血再灌注动物模型(MCAO);用甲酚紫染色法显示脑梗死区;用Western blot方法观察P-gp的表达;用ELISA法检测FK506浓度;用TUNEL法观察凋亡细胞.结果 MCAO 30 min再灌注3 h纹状体P-gp的表达开始显著升高,持续至24 h,MCAO 90 min组P-gp的表达升高幅度更大.脑缺血前后,P-gp抑制剂tariquidar(TQD)对血中FK506浓度的影响差异无统计学意义;使用TQD后,缺血侧大脑半球FK506含量显著升高.单独使用FK506时,只有较大剂量(5 mg/kg)才有抗细胞凋亡作用.单独使用TQD无抗细胞凋亡和减少脑梗死体积的作用,但当TQD与FK506合用时,FK506较低剂量(1 mg/kg)即可发挥抗细胞凋亡作用,使脑梗死体积从(113.5±11.1)mm3显著降低至(70.6±10.2)mm3(P＜0.01).结论 TQD抑制P-gp活性,增加FK506进入脑内的浓度,降低FK506脑保护作用的阈值(从5 mg/kg降低至1 mg/kg),从而加强FK506的神经保护作用.     

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.     

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.     

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.     

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.     

Treatment Considerations in Anticonvulsant Monotherapy

Summary: The long-standing practice of polypharmacy in treating epilepsy is giving way to use of monotherapy. Monotherapy can improve seizure control as well as reduce the risk of serious idiosyncratic reactions, dose-related side effects, and complex drug interactions. Monotherapy also offers improved compliance and cost-effectiveness. The basis of monotherapy is accurate diagnosis and assessment of the patient's seizure type(s), followed by selection of a single appropriate anticonvulsant drug. Many patients currently treated with multiple anticonvulsants can be successfully converted to monotherapy with a carefully monitored program in which troublesome and redundant drugs are gradually withdrawn from the therapeutic regimen.