大鼠半乳凝素9基因克隆及真核表达载体的构建

背景：研究提示,半乳凝素9在介导细胞分化、凋亡、黏附、细胞间聚集、炎症反应的调控方面发挥作用,但其作用机制尚不明确。 目的：克隆大鼠半乳凝素9基因片段,构建pDC316-GFP-Galectin-9真核表达质粒,以进一步了解半乳凝素9各种功能和反应机制, 设计、时间及地点：单一样本观察,于2008-11/2009-01在北京本元正阳基因技术有限公司完成。 材料：SPF级雄性Lewis大鼠1只。 方法：采用反转录聚合酶链反应技术,从大鼠肝脏组织中扩增出大鼠半乳凝素9基因片段,通过基因重组技术将该基因片段重组到pDC316-GFP真核表达载体上,构建pDC316-GFP-Galectin-9重组质粒,通过用PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。 主要观察指标：重组质粒的鉴定结果。 结果：总RNA经RT-PCR扩增后,经琼脂糖凝胶电泳,在1 kb下方可见一条明显扩增的条带,与预计的半乳凝素9-cDNA长度相符。重组质粒经NotⅠ/HindⅢ双酶切和未作酶切的重组质粒pDC316-GFP-Galectin-9一起经琼脂糖凝胶电泳,前者出现了980 bp和5.8 kb 2条带(980 bp为Galectin-9-cDNA产物,5.8 kb为pDC316-GFP线状质粒),后者出现6.7 kb条带(重组质粒pDC316-GFP-Galectin-9),初步表明重组质粒构建成功。测序结果用NCBI上的BLAST进行比对分析,其核酸序列与GeneBank中的半乳凝素9的mRNA的编码序列(NM_012977)同源性完全相符,进一步证实所克隆的基因为大鼠半乳凝素9-cDNA。 结论：成功克隆了半乳凝素9基因并构建成其真核表达质粒。     

重组腺病毒Ad5-mHes1的构建及其在小鼠海马组织中的表达

目的 构建含小鼠Hes1基因的重组腺病毒(rAd)并观察其在小鼠海马组织中的表达情况,为进一步研究Hes1基因在成年小鼠神经再生中的作用奠定基础。 方法 利用限制酶对pEGFP-mHes1质粒和pDC316质粒进行酶切,将获得的mHes1目的基因片段和pDC316质粒酶切产物回收,在T4 DNA连接酶作用下连接,形成pDC316-mHes1穿梭质粒,并用PCR法及EcoR Ⅰ+HindⅢ双酶切法对pDC316-mH-Hes1进行鉴定。利用AdMax包装系统,穿梭质粒pDC316-mHes1与骨架质粒pBHGlox_E1,3Cre共转染293细胞,同源重组产生复制缺陷型腺病毒Ad5-mHes1,其报告病毒是包含高强度绿色荧光蛋白(EGFP)的腺病毒Ad5 -EGFP。向成年C57BL/6小鼠海马中立体定向注射Ad5 -mHes1及Ad5-EGFP,Western blotting检测注射后7 d Hes1蛋白在海马中的表达情况,荧光显微镜观察EGFP在海马中的表达情况。 结果 用PCR法及EcoR Ⅰ+HindⅢ双酶切法对pDC316-mHes1鉴定的实验结果与预期结果相符,经测序后其序列与mHes1 CDS序列一致。注射后7d,Hes1蛋白在PBS注射组和Ad5-mHes1注射组海马组织中均有表达,其Hes1蛋白与GAPDH的比值分别为0.363±0.053和0.705±0.128,差异有统计学意义(P＜.05)。荧光显微镜下在海马齿状回颗粒细胞层中观察到Ad5-EGFP表达的绿色荧光。 结论 本研究成功构建了Ad5-mHes1表达载体系统,并在C57BL/6成年小鼠海马组织中表达了Hes1基因。     

重组腺病毒载体Ad5-hBDNF-EGFP的构建与鉴定*☆

背景：基因治疗是目前脊髓损伤治疗的方向，目的基因和载体是基因治疗的关键。 目的：构建携带增强型绿色荧光蛋白基因(Enhanced green fluorescence protein, EGFP)标志人脑源性神经营养因子(human brain-derived neurotrophic factor，hBDNF)基因重组腺病毒载体。 设计、时间及地点：单一样本观察，实验于2007-09/2008-06在福建医科大学附属第一医院完成。 材料：感受态大肠杆菌DH-5α购自美国Stratagene公司；pDC316-hBDNF、载体质粒pDC316-mCMV-EGFP、腺病毒骨架质粒pBHGlox_E1,3Cre、腺病毒包装系统AdMax和包装细胞株293购自加拿大Mixcrobix-Biosystems公司。 方法：以pDC316-hBDNF为模板，聚合酶链反应扩增酶切获得hBDNF基因片段，连接到带有EGFP标记基因的载体质粒pDC316-mCMV-EGFP上，构建穿梭质粒pDC316-hBDNF-mCMV-EGFP。利用AdMax包装系统，穿梭质粒与骨架质粒pBHGlox_E1，3Cre共转染293包装细胞, 同源重组产生复制缺陷型重组腺病毒载体Ad5-hBDNF-EGFP，反复感染293细胞扩增病毒后，离子交换法纯化病毒，并测定病毒颗粒数及滴度。 主要观察指标：①hBDNF基因原始质粒聚合酶链反应鉴定。②穿梭质粒pDC316-hBDNF-mCMV-EGFP的构建及鉴定。③重组腺病毒Ad5-hBDNF-EGFP的包装、扩增及纯化。④毒种目的基因的聚合酶链反应鉴定。⑤纯化病毒的滴度测定结果。 结果：经聚合酶链反应鉴定、限制性酶切分析及序列测定，证明已正确构建重组穿梭质粒pDC316-hBDNF-mCMV-EGFP和重组腺病毒载体Ad5-hBDNF-EGFP；扩增纯化后，测得重组腺病毒颗粒数为2.4×1011VP / mL，A260/A280值约为2.0，滴度为0.8×1010 CCID50/ mL。 结论：已成功构建重组腺病毒载体Ad5-hBDNF-EGFP，为hBDNF基因功能及基因治疗的进一步研究奠定实验基础。     

甲胎蛋白特异性小干扰RNA重组腺病毒载体的构建

背景：RNA干扰技术的应用，关键在于能够采用1个有效的基因转移系统将小干扰RNA转入至靶细胞，目前广泛应用的是构建小干扰RNA表达载体。 目的：利用AdMax腺病毒载体系统构建表达人甲胎蛋白-小干扰RNA的腺病毒载体。 设计、时间及地点：开放性实验，于2007-03/10在山东大学附属济南市中心医院中心实验室完成。 材料：穿梭质粒pDC316-EGFP-U6为本元正阳基因技术公司产品；AdMax KitD试剂盒与低代数HEK293细胞为Microbix Biosystems Inc.（Canada）公司产品。 方法：选择针对甲胎蛋白 mRNA的特异性小干扰RNA靶序列，设计合成为相应的双链DNA，并将其与酶切线性化的pDC316-EGFP-U6载体片段连接，构建好的穿梭质粒pDC316-EGFP-U6-AFP-siRNA和腺病毒骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞，同源重组产生重组腺病毒。 主要观察指标：对重组腺病毒进行聚合酶链反应鉴定及扩增、纯化、滴度测定。 结果：构建的穿梭质粒载体经聚合酶链反应鉴定和测序分析，证实与设计一致。重组腺病毒Ad-AFP-siRNA经聚合酶链反应和绿色荧光蛋白表达检测证实构建成功，测定滴度为1.4×109 nfu/L。 结论：实验成功构建了Ad-EGFP-U6-AFP-siRNA重组腺病毒。     

pcDNA3.1(+)GDNF真核表达载体的构建及其在真核细胞中的表达

目的：构建pcDNA3.1( )胶质细胞源性神经营养因子（GDNF）真核表达质粒并了解其在真核细胞内的表达。方法：将GDNF逆转录聚合酶链 式反应（RT－PCR）产物克隆至pcDNA3.1( ）真核表达载体上,经酶切鉴定及测序分析并以FuGene6介导法转染真核细胞,了解其在细胞内的表达及春表达蛋白折生物学活性。结果：RT－PCR产物为640bp特异性片段,pcDNA3.1( )GDNF重组体经酶切后分别出现640bp和300bp片段,测序分析与文献报道结果完全一致,表明重组pcDNA3.1( )GDNF表达质粒克隆成功,可见pcDNA3.1( )GDNF质粒在真核动物细胞中得到表达,GDNF蛋白能刺激含多巴胺的细胞生长,表明重组质粒能在真核动物细胞中出具有活性的GDNF蛋白。结论以FuGene6介导pcDNA3.1( )GDNF质粒传染真核细胞为基因治疗帕金森病奠定了一定基础。     

人淀粉样前体蛋白基因与绿色荧光蛋白融合基因筛选系统的建立

目的 构建携带人淀粉样前体蛋白（APP）的增强型绿色荧光蛋白（EGFP）载体。以利于初步筛选对APP mRNA水平作用的药物。方法 将pEGFP质粒和携带野生型APP695亚型的pXCJL经Hind Ⅲ酶切，连接．PCR初筛，酶切鉴定。进一步经测序证实。携带野生型人类APP751亚型cDNA的pCDNA3．1质粒用XbaⅠ酶切．Klenow酶填平．再用HindⅢ酶切．回收APP751片段；质粒EGFP用SmaⅠ和Hind Ⅲ酶切。将目的基因APP751片段与EGFP载体片段连接生成重组质粒．酶切鉴定．测序证实。构建好的重组质粒转染COS-7细胞．观察EGFP的表达情况。结果 APP695和APP751重组质粒上的EGFP在COS-7细胞内得以表达。结论 构建的APP—EGFP融合基因重组质粒．有助于方便、快速地初筛出作用于APP mRNA水平的药物。     

重组人白细胞介素1受体拮抗蛋白荧光质粒在软骨细胞的表达**☆

背景：白细胞介素1受体拮抗蛋白能延缓骨性关节炎进程,通过转基因方法可以使白细胞介素1受体拮抗蛋白表达的增加。 目的：观察重组人重组人白细胞介素1受体拮抗蛋白荧光质粒的构建及经脂质体转染软骨细胞的表达情况。 方法：双酶切法切取重组人白细胞介素1受体拮抗蛋白的c-DNA片段,通过T4DNA连接酶连接到pEGFP-C1载体上。体外分离培养兔关节软骨细胞,然后用构建的重组人白细胞介素1受体拮抗蛋白质粒经脂质体转染软骨细胞,通过荧光显微镜观察转基因的表达和荧光定量PCR检测其表达。 结果与结论：获得重组人pEGFP-C1-IL-1Ra真核表达载体质粒,酶切及测序结果证明表达质粒的DNA序列完全正确。荧光显微镜观察有绿色荧光蛋白表达,荧光定量PCR鉴定证实转染的软骨细胞基因得到表达。     

心脏转录因子GATA结合蛋白4真核表达质粒的构建*☆

背景：有研究表明心脏转录因子GATA结合蛋白4(GATA4)与先天性心脏病的发生有密切的关系,但其机制不明,而目前尚未查及GATA4真核表达质粒构建类的报道。 目的：构建人类心脏特殊转录因子GATA4的真核表达质粒。 方法：对重组质粒pUC57-GATA4进行酶切获得GATA4基因编码区序列,将真核表达载体pCMV-Myc和GATA4基因在双酶切后用T4连接酶连接,构建重组质粒pCMV-Myc-GATA4,转化至大肠杆菌,提取质粒后经聚合酶链反应、双酶切及测序鉴定。 结果与结论：实验成功酶切重组质粒获得GATA4基因编码区约1.3 kb的目的片段,聚合酶链反应和酶切鉴定以及基因测序结果显示,构建的重组真核表达质粒中含有正确的GATA4基因序列,证实成功构建了含有GATA4基因的真核表达重组质粒。     

Wilson病ATP7B基因重组腺病毒载体Ad-ATP7B的构建

目的 构建Wilson病基因ATP7B的重组腺病毒载体。方法 分别以BamHⅠ SalⅠ双酶切pcDNA3．0／ATP7B和pDC315，将ATP7BcDNA目的基因片段和线性化的pDC315连接，定向克隆构建pDC315／ATP7B，以PCR和酶切的方法鉴定。pDC315／ATP7B与腺病毒骨架共转染293细胞构建Ad-ATP7B，PCR进行鉴定。结果 经PCR和酶切鉴定证实pDC315／ATP7B构建成功。pDC315／ATP7B与腺病毒骨架共转染293细胞后见明显的毒斑，说明二者在293细胞中同源重组并包装成功。经PCR证实重组腺病毒Ad-ATP7B构建已完成。结论 本实验成功构建了ATP7B外源目的基因序列完全正确的重组腺病毒载体Ad-ATP7B，为下一步采用ATP7B基因重组腺病毒载体对Wilson病进行基因治疗打下了基础。     

保守性多巴胺能神经营养因子重组杆状病毒转移载体的构建及鉴定

目的 构建并鉴定小鼠保守性多巴胺能神经营养因子(mCDNF)重组杆状病毒转移载体pFastBacHTb-mCDNF. 方法 应用Trizol法提取小鼠组织总RNA,反转成cDNA,经PCR扩增得到带有预定酶切位点(BamH Ⅰ、Xho Ⅰ)的mCDNF基因全长(564 bp),回收片段并克隆至pGEM-T载体,测序验证PCR结果的准确性.将mCDNF定向克隆到pFastBacHTb载体,构建含有mCDNF基因的重组质粒pFastBacHTb-mCDNF,转化大肠杆菌DH5α感受态细胞,氨苄青霉素抗性筛选阳性克隆,摇菌抽取质粒进行测序和双酶切鉴定. 结果 RT-PCR扩增产物经琼脂糖凝胶电泳显示得到预定大小的目的 条带(564 bp),mCDNF的T-A克隆经蓝白斑抗性筛选获得阳性克隆,PCR及测序均提示pGEM-T-CDNF载体成功构建.重组质粒pGEM-T-mCDNF和pFastBacHTb载体进行BamH Ⅰ、Xho Ⅰ限制性内切酶酶切后再连接,得到pFastBacHTb-mCDNF重组质粒,并经PCR、酶切及测序验证无误. 结论 本实验成功构建了mCDNF重组杆状病毒转移载体pFastBacHTb-mCDNF,为该营养因子的进一步研究奠定了一定基础.     

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.