重组人胰岛素样生长因子1工程菌的高密度发酵

背景：胰岛素样生长因子1是人体内重要的细胞调控因子，已用于治疗糖尿病等多种疾病，工程菌发酵生产工艺研究是胰岛素样生长因子1实现产业化从而扩大临床应用的关键因素。 目的：探讨表达重组人胰岛素样生长因子1工程菌的高密度发酵与表达条件。 设计、时间及地点：酶基因工程实验，于2007-05/2008-05在浙江树人大学生物技术实验室完成。 材料：重组人胰岛素样生长因子1大肠杆菌表达菌株E. coli BL21(DE3)/pET22a-IGF-1由浙江树人大学生物技术实验室保存。高密度发酵补料培养基为：葡萄糖 300 g/L，蛋白胨 40 g/L，酵母粉 10 g/L，Na2HPO4 280 mmol/L，NaH2PO4•2H2O 120 mmol/L，MgSO4 10 mmol/L，氨苄青霉素100 mg/L。 方法：菌株活化后，进行摇瓶发酵试验，分别改变培养基种类、诱导剂异丙基硫代半乳糖苷浓度、诱导时间等参数，优化摇瓶发酵条件。依据摇瓶发酵优化条件，采用自控5 L发酵罐进行分批补料发酵试验。培养分分批培养和分批补料2个阶段。采用pH反馈流加的方式补料，在对数生长中后期开始诱导，加入异丙基硫代半乳糖苷至设计终浓度，培养4~6 h。 主要观察指标：重组人胰岛素样生长因子1工程菌的发酵与产物表达情况。菌体浓度与菌体干重测定，目的蛋白表达量的测定，发酵液葡萄糖浓度的测定。 结果：以2×YT＋5 g/L葡萄糖为发酵培养基，经0.8 mmol/L异丙基硫代半乳糖苷诱导5 h，通过控制溶解氧以及pH反馈补料方式，实现工程菌高密度发酵与目的蛋白高效表达，每升发酵液收获干菌体50.1 g/L ，重组人胰岛素样生长因子1含量达 5.25 g/L。 结论：实验成功建立了重组人胰岛素样生长因子1工程菌优化的高密度发酵工艺，为重组人胰岛素样生长因子1的下游纯化和工业化生产奠定了基础。     

聚乳酸/壳聚糖纤维复合支架的制备与性能

摘要 背景：聚乳酸/壳聚糖纤维复合支架材料既可提高支架的力学性能,又可中和聚乳酸的酸性降解产物,提高生物相容性,从而满足组织工程支架的要求。 目的：制备用于组织工程的聚乳酸/壳聚糖纤维复合支架。 方法：采用热致相分离法制备了聚乳酸/壳聚糖纤维复合支架。测定了复合支架的微观形貌、孔隙率、压缩模量、降解特性、蛋白质吸附特性。 结果与结论：复合支架具有纳米微米共存的亚微观结构。在聚乳酸纳米纤维网络中引入壳聚糖纤维,有效地增强了复合支架的压缩模量和蛋白质吸附能力,复合支架压缩模量为纯聚乳酸纳米支架的3.75倍,蛋白质吸附能力比纯聚乳酸纳米支架提高了112%。体外降解实验表明复合支架降解液的pH值随时间的下降明显变缓。提示,在聚乳酸纳米纤维网络中引入壳聚糖纤维,可有效增强支架的压缩模量,提高蛋白质吸附能力,并可有效减缓聚乳酸降解过程中pH值的下降,克服酸性产物引发的无痛性炎症问题。 关键词：聚乳酸;壳聚糖;纳米复合;增强;支架 doi:10.3969/j.issn.1673-8225.2010.42.015     

γ-分泌酶在神经干细胞分化过程中调节作用研究

背景：Notch信号通路在神经干细胞分化过程中起着关键作用,γ分泌酶是Notch信号通路调节的核心环节。 目的：对神经干细胞分化过程中γ分泌酶的活性、酶解产物和活性中心进行检测,为后期深入研究神经干细胞分化过程的调节机制提供一个重要的实验基础。 设计、时间及地点：观察性实验,于2008-10/2009-01在山西医科大学生物化学与分子生物学实验室完成。 材料：质粒Notch1 △E-GVP和MH100（Urban Lendahl教授惠赠）,质粒pRL-CMV（Promega公司）;15胚龄的BALB/c胎鼠。 方法：利用Gal4-VP16/UAS系统和双荧光素酶报告基因系统测定神经干细胞分化过程中γ分泌酶的活性;通过Western blot技术检测γ分泌酶酶解产物NICD的生成量;采用实时荧光定量PCR测定γ分泌酶活性中心—早老素1的表达。 主要观察指标：①双荧光素酶报告基因检测γ分泌酶的活性。②酶解产物NICD的Western blot分析。③荧光定量PCR测定早老素1的特异性表达。 结果：在γ分泌酶抑制剂DAPT作用下,γ分泌酶活性呈剂量依赖性降低,NICD的生成量也同步减少,而其催化中心组份的表达则呈反馈性的同步增高。 结论：通过对神经干细胞分化过程中γ分泌酶的活性、酶解产物和活性中心进行检测,所得结果为继续深入研究神经干细胞分化过程的调节机制奠定了坚实的前期基础。     

聚碳酸酯型聚氨酯的生物降解性

摘要 背景：不同聚氨酯材料的生物稳定性差异巨大,研究聚碳酸酯型聚氨酯的生物稳定性有利于评估其用于人工髓核外囊材料的可行性。 目的：观察聚醚型聚氨酯和具有不同硬段含量的聚碳酸酯型聚氨酯材料的体外和体内降解性能。 方法：用模拟环境进行聚氨酯的体外水解、氧化降解和酶解实验。分别置于37 ℃恒温的PBS缓冲液和“H2O2/CoCl2预氧化-Papain酶解体系”进行体外降解实验。将聚氨酯植入大耳白兔背部肌肉进行体内降解实验。 结果与结论：聚碳酸酯型聚氨酯耐降解性远优于聚醚型聚氨酯,且材料硬段含量越大,耐降解性越强。所有研究结果表明聚碳酸酯型聚氨酯具有比聚醚型聚氨酯更加优越的生物稳定性,可用作较长期人工植入生物材料。 关键词：聚碳酸酯型聚氨酯;聚醚型聚氨酯;生物稳定性;降解性;生物材料 doi:10.3969/j.issn.1673-8225.2010.47.022     

聚乳酸/海藻酸钠/壳聚糖复合材料的体外降解性能

背景：聚乳酸由于其疏水性以及在降解过程中的酸致效应，使其在应用中受到限制。通过静电组装技术在聚乳酸表面引入海藻酸钠/壳聚糖，可望克服上述缺点和不足。 目的：观察聚乳酸/海藻酸钠/壳聚糖可降解复合材料的体外降解性能。 设计、时间及地点：观察实验，于2007-09/2008-06在武汉理工大学生物中心实验室完成。 材料：采用1，6－乙二胺对聚乳酸表面进行胺解反应，形成胺化层，在其表面引入带正电的自由氨基，由静电作用依次组装上聚阴离子海藻酸钠和聚阳离子壳聚糖，获得聚乳酸/海藻酸钠/壳聚糖多层复合材料。 方法：将制备好的组装层数为5，10，15，20双层聚乳酸/海藻酸钠/壳聚糖复合材料置于37 ℃恒温的磷酸缓冲溶液中进行体外降解实验。 主要观察指标：定期测定并记录不同组装层数复合材料的pH值变化、失重及相对分子质量变化，用扫描电镜观察材料降解的形貌变化。 结果：聚乳酸/海藻酸钠/壳聚糖复合材料降解的pH值基本稳定在7.0左右；通过控制组装层数（5~15层）可有效调节材料降解过程中的pH值，pH值随层数的增加而增加。扫描电镜观察，复合材料降解7周后，材料已明显降解。 结论：聚乳酸/海藻酸钠/壳聚糖复合材料具有良好的降解性能。     

Fe3O4空心磁纳米微球制备工艺条件的影响因素

背景：空心微球材料具有很好的生物相容性，在生物医学和化学等许多领域得到广泛的应用。 目的：分析Fe3O4空心磁性纳米微球制备流程中反应温度、反应时间、洗涤次数、超声时间工艺条件对粒径的影响。 方法：以聚氧乙烯-聚氧丙烯-聚氧乙烯-嵌段共聚物F127为原料，用共沉淀法制备空心超顺磁性纳米微球。运用正交试验的数学方法设计一个4×3的正交实验模型，从反应温度、反应时间、洗涤次数、超声时间4个方面考察不同制备条件对空心磁性纳米微球粒径大小的影响。 结果与结论：得到的最佳实验制备条件：在75 ℃下，充分反应6 h，用甲醇/去离子水反复洗涤3次之后，用功率为30%的超声处理20 min。样品通过0.22 µm滤嘴过滤后，用粒径分布仪、X射线衍射和透射电子显微镜对产物进行表征观察，证实用最优的实验条件制备的空心Fe3O4磁性纳米微球粒径为37.5 nm，外壳厚度10 nm左右，颗粒大小均匀，分布较好。 关键词：工艺条件；Fe3O4；正交实验；空心纳米微球；Pluronic F-127 doi:10.3969/j.issn.1673-8225.2010.25.014     

正交优化聚乳酸-羟基乙酸纳米粒的制备

摘要 背景：聚乳酸-羟基乙酸纳米粒或纳米微球用于制备生物降解型缓释或定向给药体系已经研究了近30年,是国内外研究的热点。该体系能够控制粒径大小、延缓药物降解、延长药物释放时间、靶向释放、降低药物毒性和刺激性等。 目的：以紫杉醇为模型药物、聚乳酸-羟基乙酸为包裹材料,探索载药纳米粒的制备条件对粒径、包封率等的影响,确定最佳制备工艺条件。 方法：采用乳化-溶剂挥发法制备聚乳酸-羟基乙酸纳米粒,以粒径、包封率和载药量等为观察指标,通过正交设计法优化纳米粒制备工艺条件。 结果与结论：通过正交实验设计,优化了制备工艺条件,其最佳条件是超声乳化时间为15 min,乳化剂浓度为1%,油水相比为1∶25,合成温度为25 ℃。在此条件下进行实验,制备出的载药纳米粒粒径为217.6 nm,载药量1.79%,包封率85%。该制备工艺简单、稳定,优化制备条件,可制备出包封率高、粒径适宜的紫杉醇-聚乳酸-羟基乙酸纳米粒。 关键词：聚乳酸-羟基乙酸;紫杉醇;纳米粒;正交实验;缓释 doi:10.3969/j.issn.1673-8225.2010.42.009     

脑出血急性期D-二聚体及纤维蛋白原变化的研究

目的 :观察高血压脑出血患者急性期凝血酶时间 (Thrombin time,TT)、凝血酶原时间 (Prothrombin time,PT)、活化的部分凝血活酶时间 (Activated partial thromboplastin time,APTT)、纤维蛋白原含量 (fibrinogen,FIB)、 D-二聚体 (D- dimer)和纤维蛋白原及纤维蛋白降解产物 (fibrin- fibrinogen degradation products,FDP)的变化 ;方法 :测定5 4例急性脑出血患者入院时、 1天、 3天、 7天凝血酶时间、凝血酶原时间、活化的部分凝血活酶时间、纤维蛋白原含量、 D-二聚体、纤维蛋白原及纤维蛋白降解产物的变化。凝血酶时间、凝血酶原时间、活化的部分凝血活酶时间、纤维蛋白原含量用美国 ACL .Futura自动血凝分析仪测定。 D-二聚体采用免疫金标法 ,纤维蛋白原及纤维蛋白降解产物采用胶乳凝集法 ,试剂均由美国太平洋公司提供 ;结果 :高血压脑出血患者 3天、 7天时纤维蛋白原增高 (P<0 .0 5 ) ,D-dimer在 1天时增加 (P<0 .0 5 ) ;结论 :D- dimer暂时性升高 ,是纤溶系统时 HICH时脑组织损伤引起血中凝血活性升高的一种代偿反应。     

人重组pcDNA6.2/生长分化因子5质粒的构建及鉴定

背景：生长分化因子5是一种刺激肌腱、韧带塑型，增强愈合反应有效的生长因子，在活性组织工程肌腱构建中有重要作用。 目的：构建人重组pcDNA6.2/生长分化因子5质粒，为转染基质干细胞向肌腱诱导分化实验奠定基础。 设计、时间及地点：细胞基因工程体外实验，于2007-08/12在哈尔滨医科大学遗传学教研室和分子生物学教研室完成。 材料：根据Genbank（NM000557）中人生长分化因子5的序列化学合成一对引物,从人胎儿软骨组织提取总RNA。 方法：通过反转录-聚合酶链反应得到人生长分化因子5完整成熟肽基因。将所得基因片段插入克隆载体pcDNA6.2并转化入JM109菌株，提取重组质粒，PCR鉴定、酶切鉴定并测序。 主要观察指标：反转录-聚合酶链反应检测结果，PCR及SalⅠ和BamHⅠ双酶切鉴定结果,重组质粒测序与Genbank中序列比较结果。 结果：电泳显示，反转录-聚合酶链反应产物为一长约380 bp的带，阳性克隆质粒经PCR电泳及双酶切均出现约380 bp的片段。测序表明与Genbank中的序列完全相符。 结论：成功构建出人重组pcDNA6.2/生长分化因子5质粒，为组织工程化肌腱过程中种子细胞的制备提供转染质粒。     

亲体肝移植患者围手术期止血与凝血指标的变化

背景：亲体供肝移植后由于止血和凝血指标的异常可出现出血及血栓等严重并发症,但其具体变化规律如何尚未见报道。 目的：探讨亲体肝移植受体围手术期止血与凝血指标的变化。 方法：抽取44例亲体肝移植受者移植前、移植后1~7 d静脉血,检测患者血浆中活化部分凝血活酶时间、凝血酶原时间、凝血酶时间、纤维蛋白原含量、血小板、抗凝血酶活性、纤溶酶原活性、 纤溶酶原激活物抑制剂1活性、D-二聚体、纤维蛋白降解产物的动态变化。以32名体检中心健康体检者为对照。 结果与结论：移植前活化部分凝血活酶时间、凝血酶原时间、凝血酶时间延长(P < 0.01),纤维蛋白原含量、血小板、抗凝血酶活性、纤溶酶原活性降低(P < 0.01),纤溶酶原激活物抑制剂1升高(P < 0.01);移植后1周凝血酶原时间、活化部分凝血活酶时间、凝血酶时间、血小板、纤维蛋白原含量、抗凝血酶活性逐渐恢复正常,纤溶酶原活性、纤溶酶原激活物抑制剂1、D-二聚体、纤维蛋白降解产物仍异常于正常值(P < 0.01)。结果提示亲体肝移植后凝血因子、抗凝因子的恢复是相对较快的,而纤溶因子的恢复则较为迟缓,纤溶功能亢进,是肝脏移植严重出血的主要原因,因此在移植后凝血与止血功能的监测,是预防出血和血栓形成的有效措施。 关键词：亲体肝移植;止血;凝血;肝移植;器官移植;围手术期     

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.