C3A细胞与L-02永生化肝细胞低温保存条件下的生物学特性比较**

摘要 背景：获得大量功能良好的肝细胞是生物人工肝的核心。探索出一种可靠的肝细胞低温保存方法进而构建一个肝细胞库是目前生物人工肝研究的热点。 目的：比较用UW液在4 ℃条件下保存已经进入Ⅲ期临床试验的C3A细胞与国内构建的永生化肝细胞株L-02细胞的生物学特性。 方法：贴壁培养C3A与L-02细胞,胰酶消化,制备成细胞悬液,UW液保存。4 ℃低温保存0,24,48及72 h后,采用流式细胞术分别测定细胞存活率与凋亡率,测定谷草转氨酶与乳酸脱氢酶释放、尿素合成功能及白蛋白分泌功能。 结果与结论：随低温保存时间延长,C3A与L-02细胞存活率呈下降的趋势,但C3A细胞的存活率明显高于L-02细胞(P < 0.01);细胞凋亡率呈上升趋势,但48 h后C3A细胞同L-02细胞无差异(P > 0.05)。谷草转氨酶及乳酸脱氢酶释放呈现上升的趋势,但C3A细胞明显低于L-02细胞(P < 0.01)。白蛋白分泌功能呈下降的趋势,但C3A细胞明显优于L-02细胞(P < 0.01)。尿素合成功能呈下降的趋势,但是L-02细胞明显优于C3A细胞(P < 0.01)。结果提示,UW液4 ℃保存C3A细胞与L-02细胞时间不易超过48 h。以C3A细胞为材料的人工肝可能更适用于肝功能衰竭合并低白蛋白血症,以L-02细胞为材料的人工肝更适用于肝功能衰竭合并肝性脑病。 关键词：生物人工肝;低温保存;永生化肝细胞;C3A细胞;L-02细胞 doi:10.3969/j.issn.1673-8225.2011.03.023     

自制多脏器保存液对大鼠睾丸一氧化氮合酶的影响***

背景：在睾丸移植过程中,低温保存和缺血可导致睾丸产生氧自由基而损伤睾丸组织。 目的：观察自制多脏器保存液对低温保存大鼠睾丸一氧化氮合酶的影响。 方法：采用自制多脏器保存液和UW液低温保存大鼠睾丸,分别于保存24,48,72 h时切取睾丸,测定睾丸组织内的总抗氧化能力和一氧化氮合酶活性。 结果与结论：自制多器官保存液低温保存各时点大鼠睾丸一氧化氮合酶活性和总抗氧化能力与UW液组比较差异均无显著性意义(P > 0.05)。表明自制多脏器保存液能明显减轻低温保存大鼠睾丸氧自由基损伤,其作用与经典的美国威斯康星大学UW保存液基本相当。     

玻璃化低温保存卵巢癌细胞的配方及优化降温过程参数

实验采用玻璃化低温保存方法和程序降温方法来研究COC1细胞的低温保存效果。玻璃化低温保存：采用3种玻璃化溶液，①质量浓度为400 g/L聚乙烯基吡咯烷酮+质量浓度为200 g/L蔗糖 +质量浓度为100 g/L甘露醇。②VS55。③质量浓度为300 g/L聚乙烯基吡咯烷酮+质量浓度为200 g/L海藻糖。每种溶液以不同的比例与细胞悬液混合均匀，投入液氮保存。程序降温法：以甘油和二甲基亚砜为低温保护剂，分别以不同的比例添加到含有细胞悬液的冻存管中。然后采用两步降温法，投入液氮保存。结果玻璃化溶液（质量浓度为300 g/L聚乙烯基吡咯烷酮+质量浓度为200 g/L海藻糖）保存COC1细胞可以获得最高细胞存活率，且玻璃化方法取得的存活率高于程序降温法。初步表明，用玻璃化方法长期保存卵巢癌细胞具有一定的可行性和前景，使COC1细胞能够最大限度的保持较好的生理功能。     

丹参素干预体外低温保存大鼠肝脏血红素氧合酶1的表达

背景：研究发现丹参能够降低诱导型一氧化氮合酶mRNA 的表达，减少一氧化氮的产生，抑制肿瘤坏死因子、白细胞介素等炎性因子的分泌，具有抗氧化作用。丹参素作为丹参的重要单体成分，是否具有相同的作用? 目的：验证丹参素干预大鼠肝脏血红素氧合酶1的表达，以及对体外肝脏低温保存肝脏的保护。 方法：建立大鼠肝脏低温灌注保存模型。分为3组，对照组术前腹腔注射生理盐水，术中用乳酸林格液灌注；实验组术前腹腔注射生理盐水，术中用丹参素+乳酸林格液灌注；抑制剂组：术前腹腔注射锌原朴啉，术中用丹参素+乳酸林格液灌注。保存0，1，3，6 h，分别RT-PCR检测血红素氧合酶1mRNA及蛋白表达的情况，检测细胞线粒体钙离子含量及钙离子ATP酶活性，电镜观察各组肝细胞、线粒体形态改变，光镜观察肝细胞、肝小叶形态改变。 结果与结论：实验组肝血红素氧合酶1 mRNA及蛋白的表达水平明显比其他两组高(P < 0.05)。实验组的肝细胞线粒体钙离子含量明显较其他两组低，Ca2+-ATP酶活性较其他两组高。结果提示，丹参素灌注保存液可以诱导大鼠肝脏中血红素氧合酶1的过表达，延长肝脏低温保存时间。     

海藻糖深低温保存外周血造血干细胞的可行性

背景：二甲基亚砜是目前造血干细胞深低温保存的经典保护剂，但其对细胞和患者均有一定的毒副作用。海藻糖是一种稳定的无毒副作用的非还原性双糖，已被广泛应用于红细胞、血小板和胚胎等的冷冻保存中。 目的：探讨海藻糖作为低温保存造血干细胞保护剂的可行性。 方法：外周血造血干细胞经重组人集落刺激因子动员后，用血细胞分离机采集连续单个核细胞，分为0.5 mol/L海藻糖组、1.0 mol/L海藻糖组、对照组。采用程序降温法液氮保存，冻存7 d后取出，立即置于40 ℃水浴箱内复苏。锥虫蓝拒染法检测细胞存活率；采用甲基纤维素半固体培养体系进行集落培养，计数粒-巨噬细胞集落形成单位的回收率；采用CD34-PE/CD45-FITC双标法，流式细胞仪检测CD34+细胞回收率。 结果与结论：与对照组比较，0.5，1.0 mol/L海藻糖组细胞存活率、粒-巨噬细胞集落形成单位回收率、CD34+细胞回收率均明显升高(P < 0.001)，且0.5 mol/L海藻糖组升高幅度尤为显著(P < 0.001或P < 0.01)。证实海藻糖对于短期内低温冻存的外周血造血干细胞有一定保护作用，浓度为0.5 mol/L的海藻糖保护冻存的造血干细胞效果较佳。 关键词：低温保存；粒-巨噬细胞集落形成单位；海藻糖；浓度；外周血造血干细胞；生物材料 doi:10.3969/j.issn.1673-8225.2010.12.008     

自制PV液对大鼠肝脏低温保存的实验研究

【摘要】 目的 探讨自制PV液对大鼠肝脏低温保存的效果。方法 Wistar大鼠90只随机分成三组：UW液组、PV液组和生理盐水（NS）对照组，采用大鼠肝脏非循环离体灌注模型，每组对大鼠肝脏保存0h、6h、12h、18h、24h，每个亚组6只大鼠。测定保存不同时间段后肝脏酶学变化（ALT、AST、LDH）、灌注流出液中氧自由基代谢产物（MDA、SOD）的含量，观察胆汁分泌量及镜下肝脏形态学变化。结果 PV液组与UW液组比较，灌注流出液中ALT、AST、LDH、SOD含量相近，无显著差异（P>0.05）；保存6h后，PV液组TNF-α值较UW液组升高明显（P<0.05）；保存12h后UW液组MDA含量升高较PV液组明显（P<0.05)；保存18h后PV液组胆汁分泌量低于UW液组（P<0.05）；两组光镜、电镜下组织形态改变相似。结论 PV液与UW液对Wistar大鼠肝脏功能具有保护作用，两者短时间保存效果相似，在抗氧化及清除氧自由基方面，PV液略优于UW液。     

自制新型多器官保存液低温保存小型猪肾

背景：在前期实验的基础上,此次实验成功配制了含聚乙二醇的新型上海多器官保存液。 目的：以普通器官保存液为对照,验证自制含聚乙二醇的上海多器官保存液低温保存小型猪肾的效果。 设计、时间及地点：随机分组,对照实验观察,2006-05/2007-07在解放军器官移植研究所移植实验室完成。 材料：健康成年实验用巴马小型猪12头。自制新型新型上海多器官保存液,主要含聚乙二醇,相对分子质量20 000。pH7.43±0.10,渗透压825~900 kPa,UW液,购自美国Bristol-Myers Squibb公司。 方法：巴马小型猪肾分别以4 ℃器官保存液或上海多器官保存液原位灌注后离体保存24,48,72 h。保存终点测保存液pH值及乳酸脱氢酶含量,左肾取标本行组织学检查,右肾采用体外非循环猪肝/肾灌注系统灌注30,60,120 min测灌注液乳酸脱氢酶含量。灌注终点取肾皮质标本测丙二醛含量和超氧化物歧化酶活性。 主要观察指标：光镜、电镜观察肾冷保存后的形态学变化;灌注液及保存液乳酸脱氢酶含量;肾皮质标本测丙二醛浓度和超氧化物歧化酶活性;肾组织细胞凋亡指数。 结果：保存48,72 h后,器官保存液组保存液pH值高于上海多器官保存液组(P < 0. 05)。保存72 h终点保存液及再灌注30,60,120 min灌注液中乳酸脱氢酶活性器官保存液组高于上海多器官保存液组(P < 0.05)。同时间点两组肾组织光镜、电镜下形态学改变、肾皮质凋亡指数、超氧化物歧化酶、丙二醛含量差异均无显著性。 结论：新型上海多器官保存液对小型猪肾脏低温保存效果与普通器官保存液基本一致,对缺氧代谢器官细胞内酸中毒的缓冲能力以及对低温和缺血再灌注损伤的保护作用优于普通器官保存液。     

海藻糖对低温保存胸骨中bcl-2和bax基因表达的影响*

背景: 研究已经证实海藻糖对低温保存中的胸骨具有保护作用,但作用途径尚不清楚。 目的：观察海藻糖对低温保存胸骨bcl-2和bax基因表达的影响。 方法：新鲜配置4组保存液：低钾右旋糖酐组、低钾右旋糖酐+二甲基亚砜组、低钾右旋糖酐＋海藻糖组、低钾右旋糖酐＋二甲基亚砜组＋海藻糖组。切取SD大鼠胸骨后立即分别放入含上述4 种溶液的冻存管中低温保存。抽取新鲜大鼠胸骨组织和低温保存120 d的4组标本,采用RT-PCR方法检测不同保存液保存的胸骨及新鲜胸骨bcl-2和bax基因mRNA表达量。 结果与结论：低钾右旋糖酐+海藻糖组bcl-2表达量高于低钾右旋糖酐组、低钾右旋糖酐+二甲基亚砜组(P < 0.01),但bax表达量低于低钾右旋糖酐组、低钾右旋糖酐+二甲基亚砜组(P < 0.01);低钾右旋糖酐+二甲基亚砜组+海藻糖组bcl-2表达量最高,bax表达量最低,基本接近新鲜骨组织(P > 0.05)。结果提示海藻糖可能通过增强bcl-2基因、抑制bax基因的表达保护低温保存中的胸骨细胞活性,其与二甲基亚砜合用保护作用更好。     

新鲜人关节软骨试件在体外不同环境下退变的组织学观察

背景：采用新鲜人软骨试件是进行生物力学实验的最佳选择,但当实验不能及时完成时,何种保存方法能最大限度地保护软骨样本,使其退变达到对实验影响最小是关键。 目的：观察新鲜人关节软骨试件在体外不同环境下的组织学变化,寻找试件最佳保存方法和时间。 方法：将新鲜人关节软骨试件分为4组：正常对照组,以软骨用固定液固定;空气暴露组,暴露于空气;生理盐水组,以浸湿的生理盐水纱布包裹,滴注生理盐水;林格氏液组,以浸湿的林格氏液纱布包裹,滴注林格氏液。处理后1,2,3 h,进行苏木精-伊红染色、Masson三色染色法、番红O染色。 结果与结论：染色结果显示,生理盐水或林格氏液滴注1 h,关节软骨表面无明显改变,3 h后则出现较明显的变化,软骨表面出现明显的粗糙纤维,排列紊乱,并可见较多的裂隙。空气暴露1 h软骨表面即已出现皲裂,3 h后已失去关节软骨表面的田埂样外观,出现甚大裂隙和纤维暴露,产生软骨损伤。提示以生理盐水或林格氏液纱布包裹新鲜人离体关节软骨试件均是有效的处理方法,并且软骨保存时间最好在离体后2 h内。     

不同冷冻保护剂对低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响*★

背景：非程序降温-80 ℃低温冰箱保存方便快捷，程序降温-196 ℃液氮保存可靠长久，将两者合二为一简化流程已成功用于临床。 目的：观察不同冷冻保护剂对-80 ℃低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响。 方法：分设10%二甲亚砜组、5%二甲亚砜联合3%羟乙基淀粉组、5%二甲亚砜联合0.25 mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25 mol/L海藻糖组。采用 -80 ℃低温冰箱转液氮阶梯降温法对单采外周造血干细胞进行冷冻保存，通过透射电镜观察细胞超微结构变化，流式细胞仪观察Annexin-V、PI、Caspase-3水平。 结果与结论：4组冷冻保存细胞的存活率、凋亡率和死亡率差异均无显著性意义(P > 0.05)。透射电镜下各组细胞超微结构变化差异不明显。单个核细胞群落冷冻保存后存活率在90%以上，含成熟细胞较多的CD45+细胞群落凋亡发生率可达50%左右。造血干祖细胞群落中，早期细胞较晚期细胞更能耐受冷冻损伤。提示在基础冷冻保护剂二甲亚砜的基础上，加入羟乙基淀粉和海藻糖并未显示出对冷冻保存效果的增强作用。     

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.     

Special Pharmacokinetic Considerations in Children

Summary: Pediatric patients have greater degrees of pharmacokinetic variability and unpredictability than adults. This variability results from the effects of pharmacogenetics, age and growth, prior and current comedication, and disease. Newborns with seizures have the least predictable dosage requirements, and their needs change as drug-eliminating mechanisms mature in the neonatal period. Infants have the highest relative capacities to eliminate antiepileptics of any age group and require the largest relative doses. In addition to age-related trends, children demonstrate the same drug-specific, pharmacokinetic phenomena that adults do, including nonlinear phenytoin elimination, nonlinear valproate binding, and autoinduction of carbamazepine. Intercurrent illness and drug interactions further modify the age-related pharmacokinetic patterns in children and make dosage requirements even more unpredictable. Recent studies have shown that febrile illness can affect drug elimination, sometimes decreasing drug levels by 50% or more. Intermittent treatment with benzodiazepines administered either orally or rectally can be an important adjunct and help minimize this type of problem for children with marginally controlled epilepsy. Intermittent benzodiazepines are also helpful for children who have febrile seizures and who need only occasional antiepileptic protection.