腺体组织损伤后体外分离下颌下腺干/祖细胞后的克隆化培养**

背景：体外构建组织工程化人工涎腺需获得分化增殖良好、数量充足的种子细胞，但正常下颌下腺中很难分离出成体干细胞。 目的：利用腺体组织损伤动物模型体外分离下颌下腺干/祖细胞，并进行克隆化培养。 设计、时间和地点：细胞学体外观察，于2006-03/2007-01在遵义医学院贵州省细胞工程实验室完成。 材料：8周龄雄性SD大鼠10只，由解放军第三军医大学动物中心提供。 方法：10只大鼠采用结扎下颌下腺主导管、抑制腺体分泌来建立组织损伤模型，1周后切取腺体组织，酶消化法体外分离下颌下腺干/祖细胞，原代培养10~14 d后，挑取培养皿中形成的小的类圆形、类上皮细胞集落予以纯化，传代后进行单克隆培养。 主要观察指标：下颌下腺干/祖细胞免疫细胞化学染色及免疫荧光染色结果，通过绘制生长曲线分析下颌下腺干/祖细胞体外增殖能力。 结果：实验获得表达层粘蛋白的细胞具有干细胞特征，CD29呈阳性表达表明其具有高黏附、高增殖等组织干细胞特性，角蛋白19的阳性表达提示下颌下腺干/祖细胞呈上皮源性。其生长曲线近似“S”形，体外培养增殖活跃。 结论：实验结果显示，下颌下腺干/祖细胞具有组织干细胞的特征，有望成为组织工程化人工涎腺构建的一类种子细胞来源。     

人羊膜上皮细胞的干细胞特性***☆

背景：研究发现人羊膜上皮细胞具有类似胚胎干细胞或多能干细胞的多向分化潜能,说明其可能是未来组织工程重建的一种新型种子细胞。 目的：研究体外培养的人羊膜上皮细胞的干细胞特性。 方法：取足月剖宫产的人羊膜组织,经酶消化法和差异黏附法获得纯度高的人羊膜上皮细胞,接种于含体积分数为10%胎牛血清的DMEM/F12培养基中进行原代和传代培养,用免疫荧光法和流式细胞仪检测法检测人羊膜上皮细胞表面胚胎干细胞的表面标记蛋白OCT-4和干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达。 结果与结论：人羊膜上皮细胞在体外培养条件下呈上皮细胞特有的铺路石样外观,其胞浆中有OCT-4免疫荧光表达,其干细胞标记分子CD29、CD34的表达是阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性。结果表明人羊膜上皮细胞具有干细胞的某些特性,其可能是未来组织工程重建的一种新型种子细胞。     

胎儿心脏来源细胞的分离培养及鉴定*★

目的：胎儿心脏正处于组织器官形成和发育时期，其中的细胞数量和增殖能力应该高于成体组织，但是目前关于培养人胎儿心脏来源细胞的方法以及基本生物学特征还鲜见报道。实验拟建立体外培养胎儿心脏来源细胞的方法，探讨心脏来源细胞基本的生物学特征和细胞种类。 方法：实验于2005-10/2007-05在南京大学医学院和国家生物医药技术重点实验室完成。①实验材料：水囊引产的12~18周胎儿10例由南京大学医学院附属鼓楼医院提供，实验经产妇及家属同意，并经医院医学伦理委员会批准。②实验方法：取胎龄12~18周水囊引产的胎儿心脏，将消化后获得的细胞培养于添加内皮生长因子、碱性成纤维细胞生长因子、干细胞生长因子、心肌营养素及含10% FBS 的DMEM/F12培养基中。③实验评估：应用显微镜观察细胞的形态特征，采用RT-PCR检测培养前后心脏来源细胞干细胞标志基因等基因的表达，流式细胞仪检测培养前及培养19 d后细胞的表面标志。 结果：①心脏来源的细胞可以生长增殖。培养16 d后出现克隆，并有类似生长中心的区域形成，类似生长中心区域内的细胞较小，形态接近圆形，细胞核占细胞比例大，而周围的细胞形状多为梭形核多角形，细胞核所占的比例较小，并且显微镜下可见正在分裂的细胞。②RT-PCR检测显示，胎儿心脏组织分离获得的心肌细胞中表达c-Kit、KDR、 GATA-4、Nkx-2.5、MEF-2c，不表达c-Tnl。培养19 d后细胞中GATA-4、KDR、 MEF-2c表达量降低,不表达c-Kit，Nkx-2.5和c-Tnl。③流式细胞仪检测发现培养19 d后细胞群中CD29和CD44高表达，CD45、GATA-4和c-Tnt低表达。 结论：建立了一种体外培养胎儿心脏来源细胞的方法，并确定细胞群中有心脏来源的间充质干细胞和心肌祖细胞。     

人胎盘与骨髓源性间充质干细胞体外培养及生物学特性对比

背景：组织工程修复大块组织缺损需要高浓度、大量的细胞接种,骨髓间充质干细胞是种子细胞的主要来源,但存在数量上的局限性及长期传代后细胞功能老化的问题。 目的：体外分离培养、扩增人胎盘源性间充质干细胞与人骨髓间充质干细胞,并对其生物学性状进行比较。 设计、时间及地点：细胞学体外对照观察,于2008-09/2009-02在东直门医院实验室完成。 材料：胎盘由解放军空军总医院妇产科提供,骨髓来源于健康成人献髓者。 方法：采用密度梯度离心法从胎盘中分离、纯化和传代培养人胎盘源性间充质干细胞;骨髓肝素抗凝后,采用密度梯度离心法分离、纯化和传代培养人骨髓基质干细胞。 主要观察指标：用倒置相差显微镜观察两种细胞形态及细胞生长情况,流式细胞仪分析检测第5代细胞表面标志的表达,Von Kossa染色及油红O染色检测分化能力。 结果：镜下两种细胞均为贴壁生长,形态为均一成纤维细胞样,传5代后细胞的增殖能力随传代次数的增加而有所下降,体外培养10代后细胞生长速度减慢,但人胎盘源性间充质干细胞扩增倍数较人骨髓基质干细胞平均高出两三个数量级。两种细胞具有均一的细胞表型,均表达CD29,CD44,CD166,不表达CD34,CD45,HLA-DR。两种细胞都具有成骨、成脂分化潜能,但人胎盘源性间充质干细胞分化潜能更强。 结论：人胎盘源性间充质干细胞与人骨髓基质干细胞具有相似的生物学特性,且前者具有更强的增殖能力。     

羊水多潜能干细胞的体外培养及其生物学特性

背景：近来发现羊水中含有具有多向分化潜能的干细胞，具有向内胚层、中胚层和外胚层分化的能力。 目的：建立羊水多潜能干细胞的体外培养方法，并探讨其生物学特性。 设计、时间及地点：单一样本实验，于2007-04/2008-01在解放军军事医学科学院生物工程研究所完成。 材料：5份羊水标本来自孕16~22周流产孕妇自愿捐献。 方法：从人孕中期羊水中，采用贴壁筛选法分离羊水多潜能干细胞。 主要观察指标：采用免疫荧光、流式细胞术和反转录聚合酶链反应等方法分析细胞的生物学特性和鉴定细胞表型。 结果：体外培养的羊水多潜能干细胞呈梭形或类上皮细胞样，细胞增殖迅速，细胞倍增时间约为24h；细胞周期中G1、S、G2期的细胞所占百分比分别为72.88%、5.77%、21.35%；细胞表达Oct4、Nanog、SSEA-4、CD44和CD105，不表达CD34和CD45。 结论：采用贴壁筛选法获得的羊水多潜能干细胞增殖能力强，同时表达胚胎和成体干细胞的部分标志，可能为介于胚胎干细胞与成体干细胞之间的一种过渡阶段的干细胞。     

密度梯度离心法体外培养骨髓间充质干细胞的分化能力

背景：骨髓间充质干细胞具有多向分化能力,是目前极具潜力的种子细胞及基因治疗的靶细胞。 目的：体外培养兔骨髓来源间充质干细胞,了解其生物学特性。 方法：采用密度梯度离心法培养兔骨髓间充质干细胞,对细胞的形态及生长特性进行观察,应用流式细胞仪检测细胞表面抗原CD29、CD34、CD44、CD45、CD166、HLA-DR表达,并进行相关生物学特性鉴定。 结果与结论：密度梯度离心法能分离培养出纯度较高的骨髓间充质干细胞,流式细胞仪检测骨髓间充质干细胞表达CD29、CD44、CD166,不表达CD34、CD45、HLA-DR。在适当的条件下能够诱导分化成为成骨细胞、软骨细胞等。     

CM-Dil体外标记人脐带间充质干细胞传代示踪的可行性

背景：掌握人脐带间充质干细胞的移植示踪方法是研究其生物学特性的关键。 目的：观察用CM-Dil标记人脐带间充质干细胞及在体外传代示踪的可行性。 方法：采用酶消化法体外分离培养人脐带间充质干细胞,通过流式细胞仪检测细胞免疫表型和细胞周期、体外成脂成骨诱导鉴定该细胞。将第5代细胞用CM-Dil标记,并将细胞传代,荧光显微镜观察体外标记情况。 结果与结论：第3代人脐带间充质干细胞强表达CD44,CD29,低表达CD106,不表达CD34、CD40;有80%以上的细胞处在G0/G1期,成脂成骨诱导后,油红O染色和碱性磷酸酶染色分别阳性。CM-Dil标记人脐带间充质干细胞细胞标记率达90%以上,体外传代后荧光强度逐渐减退,传8代后,荧光基本消失。说明人脐带间充质干细胞增殖、分化能力强,CM-Dil标记细胞示踪方法简单易行。     

脂肪来源干细胞的生物学特性及细胞表型☆

背景：研究证实脂肪组织提取物中存在脂肪来源的干细胞,并表现出多向分化的潜能,但是关于其体外的生物学特性研究不够深入,其细胞表型的研究结果存在一定争议。 目的：观察脂肪来源的干细胞的生物学特性与细胞免疫表型。 方法：取吸脂手术中废弃的人脂肪组织,胶原酶消化法获得脂肪来源的干细胞,体外传代培养,并进行细胞形态学观察,细胞生长曲线测定,免疫组织化学及流式细胞仪检测其细胞表型。 结果与结论：脂肪来源的干细胞生长旺盛,呈成纤维细胞样生长,15代以内细胞形态未见明显变化;免疫组织化学染色显示,细胞表面标记CD29,CD44,CD105表达阳性,CD34,CD45表达阴性;流式细胞仪检测显示,CD29,CD44,CD105,MHC-Ⅰ为阳性,CD3,CD14,CD19,CD31,CD33,CD34,CD45,CD106,CD117,CD184为阴性。结果说明脂肪来源的干细胞体外生长能力旺盛,高表达干细胞相关抗原,可长期传代且保持低分化状态。     

组织块培养法扩增人脂肪源性干细胞的生物学特征鉴定

背景：组织块培养法分离人脂肪源性干细胞并进行体外培养,培养体系简单,节约时间,较好地保持了干细胞的生物学特性,并据此向多方向诱导分化,为组织工程学种子细胞的临床应用提供理论依据和参考路线。 目的：观察组织块培养法分离的人脂肪源性干细胞的基本生物学特性、表面抗原特点及其向成骨细胞和脂肪细胞诱导分化潜能。 方法：取健康人腹部皮下脂肪组织,组织块培养法分离出脂肪源性干细胞,进行体外培养,观察其形态特征。流式细胞仪检测第3代脂肪干细胞的细胞周期及表面抗原。取第3代人脂肪干细胞,分别用成骨和成脂诱导培养液诱导其向成骨细胞和脂肪细胞分化,Von Kossa染色、油红O染色定性鉴定。 结果与结论：原代及传代的人脂肪干细胞形态均类似成纤维细胞,生长能力旺盛。第3代人脂肪干细胞中,超过88%的细胞都处于G0/G1期。第3代脂肪干细胞表面抗原表达：CD29、CD44、CD90、CD105表达阳性,CD34、CD45、CD106表达阴性。人脂肪干细胞经成脂诱导后21 d,油红O染色阳性;成骨诱导培养后,茜素红染色和Von Kossa染色阳性。因此,实验证实人脂肪源性干细胞能向成骨细胞和脂肪细胞诱导分化。     

三维培养条件下胚胎鼠唾液腺上皮细胞的自组装**

背景：对唾液腺胚胎细胞在体外自组装及形态发生过程的理解,将有利于在体外构建唾液腺器官样组织的研究。 目的：探讨三维培养条件下SD大鼠胚胎鼠下颌下腺上皮细胞的自组装机制。 方法：将胚胎期15 d鼠的下颌下腺上皮细胞分为实验组(加入抗E-cadherin及抗β-catenin)和对照组(常规培养),在Matrigel中三维培养。 结果与结论：获得的胚胎鼠下颌下腺上皮细胞均表达CK19。实验组细胞未见分枝状及腺泡样结构出现;E-cadherin 和β-catenin荧光表达弱。对照组细胞在12 d出现上述结构;胞浆中E-cadherin、β-catenin荧光表达强表达。MOD值为实验组<对照组(P < 0.01)。提示E-cadherin,β-catenin在细胞的自组装过程中有重要作用。     

The fine structure of blood vessels in chromophobe adenoma

Summary The fine structure of the small blood vessels in chromophobe adenoma was compared to normal pituitary. The most striking difference was the relative paucity of endothelial fenestrae in the tumor accompanied by a widened perivascular space. The origin of these changes and their possible functional significance are discussed.     

The sellar barrier and intraoperative CSF leak in elderly patients

Cerebral spinal fluid (CSF) leak is a significant complication in pituitary surgery, increasing both patient morbidity and mortality. In a recent publication, Campero et al. observed worse postoperative prognosis and increased risk of intraoperative CSF leak in patients with reduced sellar barrier thickness. The objective of this study was to analyze the association between sellar barrier thickness and intraoperative CSF leak in older individuals. A retrospective review was conducted of 44 transsphenoidal surgery resections for pituitary adenomas, 24 microscopic and 20 purely endoscopic procedures. Presence of intraoperative CSF fistula was significantly greater in patients with weak sellar barrier (thickness under 1 mm), compared to strong sellar barrier (52.94% vs 3.70% p < 0.0001, respectively). Application of this novel concept may help improve surgical technique selection as well as predict risk of intraoperative CSF leak and need for eventual use of flaps for reconstruction.     

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 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.     

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.     

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.     

Anticonvulsant Drugs and Cognitive Function: A Review of the Literature

Summary: Alterations of cognitive function are separate from disturbances of behavior seen in association with epilepsy. The nature of the cognitive disability may to a certain extent depend on the seizure type. Partial seizures, mainly derived from a temporal lobe focus, impair memory tasks, while generalized seizures seem to have more effect on attentional abilities. A number of studies, reviewed in this paper, suggest that anticonvulsant drugs further impair cognitive function. Maximal impairments are seen in patients receiving polytherapy: rationalization of polytherapy improves cognitive abilities. Studies in children and adults have allowed differentiation of the effects of various commonly used antiepileptic agents. Maximal cognitive deficits are seen with. phenytoin, while phenobarbital and sodium valproate induce moderate disturbances, and carbamazepine seems relatively free from such toxicity. Further research is needed on the interrelationship between types of seizure disorders, types of anticonvulsant medications, and cognitive function.     

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.