MC3T3-E1细胞在改性聚（DL-乳酸）表面的黏附性能*★

背景：聚乳酸是经FDA批准可进入人体的生物可降解材料，因其表面疏水性强，与细胞亲和力差，所显示的生物相容性较差。 目的：观察MC3T3-E1成骨前体细胞在马来酸酐改性聚乳酸和丁二胺改性聚乳酸表面的黏附情况，评价改性聚乳酸的细胞相容性，并与聚（DL-乳酸）对比。 设计、时间及地点：对比实验，于2007-06在重庆大学生物工程学院生物材料与仿生工程研究中心完成。 材料：聚（DL-乳酸）、马来酸酐改性聚乳酸和丁二胺改性聚乳酸为自制；MC3T3-E1细胞株购自上海细胞生物研究所。 方法：采用体外培养细胞的方法，将MC3T3-E1细胞直接接种到聚（DL-乳酸）、马来酸酐改性聚乳酸和丁二胺改性聚乳酸材料上。 主要观察指标：通过细胞形态、细胞增殖、细胞周期、细胞黏附检测比较不同基底材料对MC3T3-E1成骨前体细胞的影响。 结果：MC3T3-E1成骨前体细胞在马来酸酐及丁二胺改性聚乳酸膜上的增殖速率和黏附力大于聚（DL-乳酸）(P < 0.05)；细胞周期测定显示马来酸酐及丁二胺改性聚乳酸膜能促进MC3T3-E1成骨前体细胞从G0期向S期和G2/M期过渡，细胞形态也较为成熟。 结论：马来酸酐改性的聚乳酸和丁二胺改性的聚乳酸能促进MC3T3-E1成骨前体细胞黏附、增殖，并促进其更迅速地从成骨前体细胞向成骨细胞分化，具有比聚乳酸更好的细胞相容性。     

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。     

整合素α5β1在成骨细胞与生物衍生材料黏附过程中的表达

背景：整合素在细胞与材料的黏附过程中发挥重要作用。 目的：了解整合素α5β1在成骨细胞构建组织工程骨和组织工程骨膜过程中的表达,探讨在成骨细胞与生物衍生材料黏附过程中整合素α5β1发挥的作用。 方法：选用人胚骨膜来源成骨细胞为种子细胞,接种生物衍生骨及羊膜支架材料上,制备组织工程骨及骨膜,分别培养2,4,6,8,10 d。单纯培养的成骨细胞作为对照组。 结果与结论：实时定量PCR测定结果,培养早期整合素α5表达呈阴性,整合素β1组织工程骨中的表达略高于组织工程骨膜,培养2,6 d组比较差异有显著性意义。单纯细胞培养的对照组中,整合素β1稳定,各时间段比较差异无显著性意义。提示生物衍生材料有利于成骨细胞黏附,但在黏附过程的初期,成骨细胞可能是通过纤维连接蛋白以外的其他蛋白质完成该过程的。     

肿瘤坏死因子α干预小鼠成骨细胞cbfa1/runx2基因的表达*

背景：肿瘤坏死因子α可降低牙周膜纤维细胞碱性磷酸酶的活性,抑制牙周膜纤维细胞向成骨细胞的功能转化。 目的：观察肿瘤坏死因子α对小鼠成骨细胞生长及cbfa1/runx2基因表达的影响。 方法：取生长良好的小鼠成骨细胞系MC3T3/E1细胞,分别以20,40,60,80 μg/L的肿瘤坏死因子α进行干预,以正常培养的细胞作为对照。采用RT-PCR法检测MC3T3/E1细胞cbfa1/runx2 mRNA的表达;PNPP法测定碱性磷酸酶活性;MTT法检测细胞活力。 结果与结论：正常培养的MC3T3/E1细胞cbfa1/runx2 mRNA呈阳性表达,随着肿瘤坏死因子α浓度的增高,其表达水平逐渐下降。同时MC3T3/E1细胞活力和碱性磷酸酶活性也随肿瘤坏死因子α浓度的增高而下降。提示肿瘤坏死因子α可抑制MC3T3/E1细胞生长,而cbfa1/runx2可能参与了成骨细胞的分化过程。     

可降解镁锌合金的细胞毒性和溶血实验

背景：自主设计的可降解镁锌合金既保持了镁合金与人骨接近的密度和弹性模量，又排除了商业镁合金中铝和稀土元素的毒性。 目的：评价自制可降解Mg-6wt%Zn合金的细胞相容性。 设计、时间及地点：对比观察实验，于2007-11/2008-03在上海交通大学附属第六人民医院中心实验室完成。 材料：可降解Mg-6wt%Zn合金由上海交通大学材料科学与工程学院研制，密度1.82 g/cm3，弹性模量44 GPa。细胞毒性实验所用L-929细胞由中国科学院典型培养物保藏委员会细胞库提供。溶血实验所用血取自成年雄性新西兰大白兔。 方法：将Mg-Zn合金浸提液稀释成100%，50%，10%，加入96孔板里培养L-929细胞，并设单纯的DMEM培养液为阴性对照组，加入0.64%苯酚的DMEM培养液为阳性对照组。 主要观察指标：分别在第2，4，7天用MTT比色法定量测试材料对L-929细胞相对增殖率的影响, 并根据ISO 10993-5:1999标准评估细胞毒性。应用倒置显微镜观察细胞培养2，4，7 d 后形态和生长状况。按照ISO 10993-4:2002标准，通过对材料与兔血红细胞在体外接触过程中，所致红细胞溶解和血红蛋白游离程度的测定，评价材料的体外溶血性。 结果：随着培养时间的增加，细胞形态保持正常，数量明显增多，不同浓度镁锌浸提液中细胞生长形态与阴性对照并无明显差异。可降解镁锌合金对L-929细胞无明显毒性作用，毒性评价为0~1级。可降解镁锌合金对血液的溶血率为3.4%，小于标准规定的5%，属于无溶血反应。 结论：可降解镁锌合金无明显细胞毒性，细胞相容性较好。     

两种磷酸钙骨水泥浆料固化过程对成骨细胞的影响

背景：国内外关于骨水泥浆料固化过程中对细胞影响及解决方案的研究报道甚少。 目的：观察β-磷酸三钙/磷酸钙骨水泥和壳聚糖微球/磷酸钙骨水泥浆料固化过程对成骨细胞的影响。 设计、时间及地点：对比观察,于2006-08/12在北京大学医学部细胞与生物研究室完成。 材料：自制2种可注射磷酸钙骨水泥：β-磷酸三钙/磷酸钙骨水泥和壳聚糖微球/磷酸钙骨水泥;小鼠成骨前体细胞株MC3T3-E1由日本RIKEN细胞库提供。 方法：将小鼠成骨前体细胞株MC3T3-E1分别接种于β-磷酸三钙/磷酸钙骨水泥和壳聚糖微球/磷酸钙骨水泥的固化块及浆料上,以细胞接种于24孔板作空白对照,用吖啶橙染色后在荧光显微镜下观察,对细胞进行计数分析。 主要观察指标：①细胞形态。②细胞计数。 结果：①荧光显微镜下可见黏附于材料块上活细胞的核呈亮绿色荧光,细胞形态完整,未见裂解。材料也吸收部分荧光,呈深绿色的背景。而在磷酸钙骨水泥浆料上仅有少数活细胞存留,明显少于β-磷酸三钙/磷酸钙骨水泥固化块组和空白对照组。②β-磷酸三钙/磷酸钙骨水泥和壳聚糖微球/磷酸钙骨水泥固化块与空白对照组相比,对MC3T3-E1细胞数无影响。两种材料浆料上的细胞数显著低于相应骨水泥固化块组和空白对照组(P < 0.01)。 结论：β-磷酸三钙/磷酸钙骨水泥和壳聚糖微球/磷酸钙骨水泥材料的浆料固化过程对MC3T3-E1细胞有不利影响,致使细胞数降低。     

雌激素受体基因ERβ与小鼠成骨细胞的增殖和分化

背景：雌激素受体ERs表达于所有关系到骨形成和骨吸收的细胞成分中。 目的：观察雌激素受体ERβ对成骨细胞增殖、分化能力的调控作用。 方法：以小鼠成骨细胞株MC3T3-E1为对象,设立3组：实验组转染雌激素受体ERβ RNAi载体、阴性对照组转染ERL RNAi载体、空白对照组不进行转染,3组在相同条件下培养。采用MTT法绘制各组细胞的生长曲线;流式细胞仪分析各组细胞的细胞周期。 结果与结论：实验组成骨细胞的增殖能力明显高于空白对照组和阴性对照组,G1期细胞百分率明显少于空白对照组和阴性对照组,而S期和G2期细胞百分率明显高于空白对照组和阴性对照组(P均< 0.05)。根据雌激素受体ERβ沉默后检测的结果可以反向推断,ERβ的表达对成骨细胞的增殖、分化具有抑制作用。     

两种新型支架材料的细胞相容性

背景：利用骨组织工程原理,通过观察成骨细胞在生物材料上的形态、测定成骨细胞在生物材料上的黏附率等方法,可评估生物支架材料的生物相容性。 目的：通过扫描电子显微镜下细胞-生物材料的观察和细胞-生物材料黏附率的测定,探讨骨组织工程材料的细胞相容性。 设计、时间及地点：动物实验,成骨细胞形态学观察,于2007-02/2007-09在华南理工大学材料科学与工程学院生物材料研究所教育部重点实验室完成。 材料：健康 SD乳鼠30只,2种支架材料分别是：复合海藻酸钠和聚乳酸羟基乙酸的磷酸钙骨水泥以及卵磷脂改性聚乳酸羟基乙酸/生物活性玻璃复合支架。 方法：取SD乳鼠颅骨成骨细胞,经体外培养、扩增,与两种新型可降解生物活性支架材料进行复合,通过扫描电镜观察细胞形态并测定细胞在生物材料上的黏附率。 主要观察指标：观察接种于生物材料表面上的成骨细胞形态、黏附情况,对生物材料生物相容性进行描述。 结果：生物活性材料与细胞复合后,材料出现了矿化,同时在海藻酸钠/聚乳酸羟基乙酸磷酸钙骨水泥通过扫描电镜观察发现其上游生长形态良好的成骨细胞;成骨细胞在生物卵磷脂改性聚乳酸羟基乙酸生物活性玻璃上的黏附率最高达到了95.2%。 结论：从细胞形态和细胞黏附率上看,海藻酸钠/聚乳酸羟基乙酸磷酸钙骨水泥和生物卵磷脂改性聚乳酸羟基乙酸生物活性玻璃具有较好的生物相容性。     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示，小分子肽能明显增加去卵巢大鼠的骨钙含量，使其骨密度增加，能很好地预防骨质疏松。同时体外实验显示，小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化，并且可能是通过抑制核转录因子 p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。 目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。 方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组，50，100 mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1，分别于作用3，6，12，18，24，30 d后，收集细胞提取蛋白，Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。 结果与结论：50，100 mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达(P < 0.01)，而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组(P < 0.01)。因此，认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统，间接地抑制破骨细胞的数量和功能。     

健骨颗粒含药血清培养成骨细胞整合素/黏着斑激酶信号通路相关因子的 表达**☆

背景：目前有关成骨细胞整合素的功能状态及整合素信号转导调控的研究较少。健骨颗粒对成骨细胞整合素表达水平及信号转导通路调控的作用未被阐明。 目的：观察健骨颗粒含药血清对成骨细胞黏附的影响，揭示健骨颗粒对成骨细胞整合素表达水平及信号转导通路的调控作用。 方法：健康雄性3月龄SD大鼠用于制备健骨颗粒含药血清，选取最佳剂量的含药血清干预第3代大鼠成骨细胞，ELISA法检测培养液中纤连蛋白、Ⅰ型胶原、玻连蛋白的水平，Western blot法检测成骨细胞黏着斑激酶的磷酸化情况，实时荧光定量PCR法检测成骨细胞纤连蛋白、Ⅰ型胶原、玻连蛋白、黏着斑激酶mRNA的表达。 结果与结论：浓度为20%的健骨颗粒含药血清能明显增强体外培养成骨细胞的增殖活力。随着含药血清干预时间的延长，成骨细胞表达及分泌的纤连蛋白、Ⅰ型胶原、玻连蛋白、黏着斑激酶mRNA及蛋白水平逐渐上升，黏着斑激酶的磷酸化水平也逐渐增高(P < 0.05)；均明显高于同期的生理盐水血清干预水平(P < 0.05或P < 0.01)。说明健骨颗粒能作用于成骨细胞整合素激活的粘着斑激酶转导通路各相关因子，使成骨细胞进入“分泌增加→黏附增加→功能增强、分裂增殖→分泌增加”的良性循环，对成骨细胞的成骨效应起正性调节作用。 关键词：健骨颗粒；成骨细胞；整合素；粘着斑激酶；骨质疏松症     

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.