大鼠成骨细胞的原代培养和鉴定

背景：组织工程需要大量的种子细胞,成骨细胞已成为骨组织工程构建的重要种子细胞之一。但成骨细胞取材困难,获得的成骨细胞的纯度不一。 目的：建立大鼠新生乳鼠颅骨来源成骨细胞的分离培养纯化方法,观察颅骨来源成骨细胞生物学特点。 方法：采用二次酶消化法对SD大鼠乳鼠成骨细胞进行原代培养,扩增。通过差速贴壁法进行成骨细胞纯化。通过形态学、细胞碱性磷酸酶检测、茜素红染色、钙结节Von kossa法染色、超微结构以及细胞增殖曲线,确定其增殖与成骨活性。 结果与结论：二次酶消化法培养颅骨来源成骨细胞可获得原代细胞增殖,传代扩增细胞具有典型成骨细胞形态学和生物学活性。碱性磷酸酶、茜素红染色、钙结节Von kossa法染色均呈阳性结果。超微结构显示为高分化功能活跃成骨细胞,细胞增殖曲线显示细胞生长活跃。提示,新生SD大鼠颅骨来源成骨细胞具有良好的增殖与成骨活性,能连续传代增殖,纯度高,细胞生物学特征稳定,适用于做体外实验研究。     

人胚胎骨骨膜来源成骨细胞的分离培养与生物学特性

背景：胚胎骨来源成骨细胞因其低免疫原性和高增殖与成骨活性有可能成为适用于骨组织工程方法异体骨组织缺损修复的种子细胞。冻存复苏后的生物学特点是否能进行进一步临床应用研究有待证实。 目的：建立人胚胎骨骨膜来源成骨细胞的分离培养、保存方法,观察骨膜来源成骨细胞生物学特点。 设计、时间及地点：细胞形态学与生物学特性实验研究,于2005-04/2006-05在天津市口腔医院细胞与组织工程实验室完成。 材料：细胞来源为5月龄自然流产胎儿,产妇及家属知情同意,无菌条件下取长骨骨膜组织。 方法：采用组织块法对成骨细胞进行原代培养,传代培养细胞液氮冻存3~6个月后,选择不同代次细胞复苏培养。 主要观察指标：通过形态学、超微结构,细胞增殖曲线,钙结节Von kossa法染色以及细胞内碱性磷酸酶定量检测与钙钴法染色确定其增殖与成骨活性。 结果：组织块法培养骨膜来源成骨细胞第6天即可获得原代细胞增殖,传代扩增细胞具有典型成骨细胞形态学和生物学活性。苏木精-伊红染色密集融合的成骨细胞与细胞外基质显示转化为脊样骨组织结构排列。经统计学分析定量检测成骨细胞碱性磷酸酶活性与细胞密度有线性正相关关系。液氮冷冻保存后复苏培养复层成骨细胞钙钴法碱性磷酸酶染色阳性率在45%以上,超微结构显示为高分化功能活跃成骨细胞。 结论：胚胎骨骨膜来源成骨细胞具有良好的增殖与成骨活性。深低温液氮冻存成骨细胞复苏后仍能连续传代增殖并具有良好的成骨代谢活性,能够体外借助细胞外基质形成骨样结构。     

聚乳酸-壳聚糖纤维/羟基磷灰石-硅酸钙复合支架材料的细胞相容性

背景：实践证明,有机和无机材料单独应用都不是理想的支架材料。聚乳酸具有良好的生物相容性、生物降解性和生物吸收性,聚乳酸类复合材料将成为21世纪最重要的生物复合材料之一。 目的：观察复合支架材料聚乳酸-壳聚糖纤维/羟基磷灰石-硅酸钙对成骨细胞黏附、增殖、分化的影响。 方法：取新生24 h内Wistar大鼠的颅盖骨,采用改良胶原酶消化法进行成骨细胞原代培养。通过倒置相差显微镜、苏木 精-伊红染色、碱性磷酸酶染色、钙结节茜素红染色对获得的细胞进行生物学特性的观察与鉴定。然后将第3代细胞与聚乳酸/壳聚糖纤维、聚乳酸-壳聚糖纤维/硅酸钙、聚乳酸-壳聚糖纤维/羟基磷灰石-硅酸钙三种支架材料体外复合培养。培养3,6,9 d后,采用倒置相差显微镜观察材料周边的细胞形态,通过碱性磷酸酶活性测定法和MTT法观察3种支架材料对细胞分化、增殖的影响。 结果与结论：三种材料均有利于成骨细胞的黏附、生长、分化、增殖,而聚乳酸-壳聚糖纤维/羟基磷灰石-硅酸钙复合支架材料较聚乳酸/壳聚糖纤维、聚乳酸-壳聚糖纤维/硅酸钙支架材料促进成骨细胞的分化增殖效果更好,证实其生物相容性好,有望成为一种新型的骨组织工程支架材料。 关键词：复合支架;细胞相容性;生物材料;成骨细胞;组织工程支架材料;骨组织工程 doi:10.3969/j.issn.1673-8225.2010.08.016     

体外冲击波对大鼠成骨细胞增殖、分化、黏附及迁移的影响

背景：体外冲击波是治疗骨折延迟愈合或不愈合的一种有效方法,成骨细胞在此过程中发挥着重要的作用。 目的：研究成骨细胞在体外冲击波促进骨折愈合过程中的作用,为提高冲击波治疗效果提供理论支持。 方法：将原代培养的SD大鼠成骨细胞随机分成冲击波组和对照组,应用不同能量的冲击波进行处理后接种于96孔培养板,根据细胞存活率和细胞增殖情况确定适宜的冲击波能量值。钙钴法染色观察成骨细胞碱性磷酸酶,CCK-8试剂盒检测细胞存活,AKP试剂盒检测AKP表达,茜素红染色观察矿化结节,流式细胞仪检测整合素β1及RT-PCR检测整合素β1 mRNA表达,伤口愈合试验观察成骨细胞的迁移率。 结果与结论：冲击波处理体外原代培养成骨细胞的适宜能量为10 kV(500脉冲),冲击波组细胞增殖速度快,细胞分泌碱性磷酸酶水平高,矿化结节面积大,细胞黏附率高,整合素β1及其mRNA的表达均高于对照组 (P < 0.01),且冲击波组细胞迁移的平均距离大于对照组(P < 0.05),提示适宜能量冲击波可促进成骨细胞的增殖、分化、黏附及迁移,同时,整合素β1在细胞黏附及迁移过程中可能扮演了重要的角色。     

胶原-纳米羟基磷灰石复合支架的细胞相容性

背景：观察成骨细胞在生物材料上的形态、增殖和分化等项目,可评估生物支架材料的生物相容性。 目的：观察复合支架材料纳米羟基磷灰石/胶原对成骨细胞增殖、分化的影响。 方法：取新生24 h内Wistar大鼠的颅盖骨,采用改良胶原酶消化法进行成骨细胞原代培养,取第3代细胞与纳米羟基磷灰石/胶原支架或普通羟基磷灰石材料体外复合培养。培养3,6,9 d后,观察材料周边的细胞形态及支架材料对细胞分化、增殖的影响。 结果与结论：纳米羟基磷灰石/胶原材料较普通的羟基磷灰石材料更有利于成骨细胞的黏附、生长、分化、增殖,证实其生物相容性更好,有望成为一种新型的骨组织工程支架材料。     

聚L-乳酸/聚L－乳酸接枝的羟基磷灰石复合材料（PLLA/PLLA-gHAP）对成骨细胞黏附和増殖能力的影响

目的：观察新型骨修复材料聚L－乳酸/聚L－乳酸接枝的纳米羟基磷灰石（PLLA/PLLA-gHA）对成骨细胞黏附、增殖功能的影响,为其今后的研究和应用提供依据。 方法：实验于2006-3/2007-3在东北师范大学细胞与遗传研究所完成。取孕19~20天的Wistar大鼠胎鼠的颅骨进行成骨细胞体外培养。①光镜下观察成骨细胞形态,应用碱性磷酸酶染色、钙结节染色对其进行鉴定。②将成骨细胞分别种在PLLA/PLLA-gHA和PLLA材料板上,24小时后应用细胞技术器检测细胞黏附数量,扫描电镜观察细胞形态③MTT法测定成骨细胞在不同浓度（10g/L,100g/L）的PLLA/PLLA-gHA材料浸提液中的増殖情况,并与PLLA材料进行对比。 结果：①成骨细胞特性观察：光镜下成骨细胞呈梭形或多角形生长,碱性磷酸酶染色及茜素红钙结节染色阳性。②在PLLA/PLLA-gHA表面黏附的细胞数略多于PLLA材料,且粘附于PLLA/PLLA-gHA材料表面的成骨细胞直径较大,伸出的伪足与材料间形成一种牢固的锚状结构。而在PLLA表面的细胞细胞直径较小,多为球形,很少伸出伪足。③10g/L PLLA/ PLLA-gHA浸提液组的细胞活性明显高于10g/LPLLA浸提液组和空白对照组;两种材料100g/L浓度组的细胞活性均低于空白对照组,但PLLA/ PLLA-gHA组的细胞活性仍高于PLLA组。 结论：①成骨细胞在PLLA/PLLA-gHA材料表面较PLLA材料表面显示更好的黏附能力。②成骨细胞在PLLA/PLLA-gHA材料浸提掖中较在PLLA材料浸提掖中显示更好的増殖能力。③新型PLLA/PLLA-gHA材料具有较好的成骨细胞相容性,可作为骨修复材料使用。     

骨关节炎软骨细胞的体外分离培养

背景：由于体外分离培养骨关节炎患者软骨细胞存在难度，同时软骨组织中因富含大量的基质, 且软骨细胞分布于其中的软骨陷窝中, 因此与其他细胞相比, 软骨细胞的分离更为困难。 目的：从骨关节炎行人工膝关节置换者的关节软骨中分离软骨细胞，并改良其酶消化法分离软骨组织，观察软骨细胞的生物学特性。 设计、时间及地点：对比观察，实验于2007-04/2008-03在解放军第三军医大学烧伤研究所实验室完成。 对象：标本取材于解放军第三军医大学西南医院关节外科中心同期收治的10例人工膝关节置换患者软骨组织（> 3 cm）。 方法：切取原发性骨关节炎患者关节软骨，将软骨剪成1 mm×1 mm×1 mm左右的碎块，以Ⅱ型胶原酶顺序消化联合胰蛋白酶消化分离关节软骨细胞,同期对比用Ⅱ型胶原酶加胰蛋白酶消化法消化软骨后的收获细胞数及细胞成活率，分离细胞进行原代和传代培养。 主要观察指标：以倒置相差显微镜观察体外培养软骨细胞形态；以甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学染色进行软骨细胞鉴定；四甲基偶氮唑盐法测定骨关节炎细胞增殖情况。 结果：改良的Ⅱ型胶原酶顺序消化联合胰蛋白酶法，对原代关节软骨细胞的分离取得了优良而稳定的效果，与传统的胰蛋白酶消化法相比，收获细胞数为3.72×106，细胞存活率为97.5%，二者相比有显著性差异（P < 0.05）。体外培养观察软骨细胞贴壁和生长均较缓慢。原代和传代后细胞Ⅱ型胶原免疫组织化学和甲苯胺蓝异染反应均较弱。软骨细胞增殖和生长缓慢。 结论：Ⅱ型胶原酶顺序消化联合胰蛋白酶分离骨关节炎患者软骨细胞的技术简单且易操作, 分离培养的软骨细胞符合骨关节炎时软骨退变的表现, 可为骨关节炎的病因研究及软骨细胞移植治疗骨性关节炎提供实验基础。     

改良分步消化法培养原代软骨细胞**★

背景：胰蛋白酶和细菌胶原酶结合使用消化关节软骨基质获得大量纯度高的软骨细胞的方法步骤繁琐、过程复杂，容易污染，但更好的简单易行、安全可靠的方法至今少有报道。 目的：采用改良分步消化法进行软骨细胞培养，以获取大量纯净的软骨细胞。 方法：将新西兰白兔6只随机分2组，酶消化法组运用酶消化法分两步获取原代软骨细胞，对照组用传统法进行原代软骨细胞培养。培养1周后观察两组培养的软骨细胞的生长状态，并进行细胞鉴定、计数，评估改良后的方法对细胞的影响。 结果与结论：酶消化法组采用0.2%Ⅱ型胶原酶消化软骨细胞，与对照组相比，可将6 h以上的消化时间缩短至3 h。两组原代软骨细胞培养24 h均多呈圆形，悬浮状态，48 h后贴壁，培养1周后，两组软骨细胞可铺满培养瓶底。结果证实，采用改良分步消化法进行软骨细胞培养，在缩短了消化时间的同时其细胞生长及形态变化均无改变，可以顺利获取大量纯净的软骨细胞。     

胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞*

背景：近年来，众多研究欲采用体外分离培养的关节软骨细胞作为修复缺损的关节软骨的种子细胞，然而，获得纯化的具有生物活性的关节软骨细胞较为困难。 目的：拟运用胰蛋白酶与Ⅱ型胶原酶联合消化获取关节软骨细胞。 方法：从SD大鼠的正常股骨及胫骨关节表面获取关节软骨，先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化，显微镜下见大量细胞游离后，弃去大块的未消化的关节软骨碎片，离心，去上清，PBS洗涤2次后，加入软骨细胞原代培养液进行培养、增殖。应用甲苯胺蓝及苏木精-伊红染色方法检验所得细胞是否为关节软骨细胞。 结果与结论：在严格掌握酶的浓度及消化时间的前提下，通过0.25%胰蛋白酶和0.2%Ⅱ型胶原酶联合酶解关节软骨的方法，成功从大鼠股骨及胫骨关节软骨内分离培养出细胞，并经过甲苯胺蓝染色及苏木精-伊红染色证实，所得的细胞为具有生物活性的关节软骨细胞。 关键词：关节软骨细胞；Ⅱ型胶原酶；胰蛋白酶；联合消化；甲苯胺蓝；苏木精-伊红染色     

体外培养骨髓基质细胞向成骨细胞的分化

背景：目前如何有效地诱导骨髓基质细胞向成骨细胞转化，建立稳定的体外培养诱导模式，研究诱导条件的作用机制是骨组织工程研究的重点。 目的：建立一种兔骨髓基质细胞向成骨细胞分化的体外培养体系，观察其成骨过程。 设计：对比观察。 材料：4~8周龄新西兰大白兔，雌雄不拘，用于骨髓基质细胞的原代与传代培养。 方法：将兔股骨骨髓基质细胞进行原代培养，待细胞铺满瓶底近80%后，加入2.5 g/L胰蛋白酶消化传代培养。取生长良好的对数期第2代细胞，以1×108 L-1的细胞浓度接种于预置盖玻片的6孔培养板内。细胞分为2组，实验组使用条件诱导培养基(RPMI 1640标准培养基加入地塞米松10-8 mol/L，β-甘油磷酸钠10 mmol/L，维生素C 50 μg/L)，对照组继续使用标准培养基。两组细胞持续培养(常规两三天换液1次)进行细胞成骨分化观察。 主要观察指标：倒置相差显微镜观察细胞生长及形态变化；苏木精-伊红染色、碱性磷酸酶染色、Ⅰ型胶原免疫组织化学染色、钙结节染色及扫描电镜观察骨髓基质细胞形态变化。 结果：经诱导培养的细胞，在体外逐渐转化、增殖、分泌细胞外基质、最终形成了矿化基质。苏木精-伊红染色显示实验组传代细胞在接近融合为单层时形态多为梭形，多角形等。胞浆内可见颗粒；碱性磷酸酶染色可见实验组传代细胞染色呈强阳性，阳性率达83.2%，并能大量分泌Ⅰ型胶原；对照组仅有个别细胞染色呈阳性。四环素染色观察实验组骨髓基质细胞形成金黄色钙结节，甚至是骨样组织，与典型成骨细胞的生物学特性相似。扫描电镜观察实验组培养的传代细胞已接近融合为单层，细胞呈梭形或多角形表现，细胞表面及细胞间可见钙盐结晶沉积。 结论：实验所建立的兔骨髓基质细胞体外诱导成骨转化体系，能够培养出生长活跃，增殖迅速，稳定、多量向成骨细胞转化的骨髓基质细胞培养模式，所培养的细胞可作为成骨细胞的一种来源，为构建组织工程化骨提供种子细胞。     

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.     

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.     

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.     

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.     

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.     

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.