SNCA基因过表达慢病毒质粒构建及稳定转染293T细胞系

目的构建SNCA基因过表达慢病毒质粒,转染293T细胞,建立稳定转染细胞系。方法应用PCR技术扩增目的基因,并将扩增产物插入慢病毒载体质粒pGC-FU上,并对阳性克隆进行基因测序鉴定。pGC-FU-SNCA-GFP重组质粒包装293T细胞,转染24小时后,用荧光显微镜观察标签GFP绿色荧光蛋白的表达,并用West blotting法测定目的蛋白的表达。结果成功构建了pGC-FU-SNCA-GFP慢病毒过表达质粒,获得了稳定转染的293T细胞株。结论人SNCA基因过表达慢病毒载体成功,构建和稳定转染293T细胞系的建立,为进一步体外研究α-突触核蛋白的功能奠定了基础。     

Nurrl基因在大鼠体外培养骨髓基质细胞的表达

目的:为获得高效表达孤儿核受体(orphan nuclear receptor,Nurrl)基因的骨髓基质细胞.方法:采用分子克隆技术,构建携带Nurrl基因的重组腺相关病毒(adeno-associated virus,AAV)载体;与包装质粒pAAV-RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK293制备具有感染活性的AAV-Nurrl病毒粒子;用病毒上清液感染原代培养的大鼠骨髓基质细胞(Marrow stromal cells,MSCs),并用免疫细胞化学方法检测阳性细胞.结果:经酶切鉴定和DNA测序,得到了序列正确的重组pAAV-Nurrl;感染HT1080细胞进行病毒滴度测定,每mL病毒贮存液可达1012个阳性细胞;获得了Nurrl阳性的骨髓基质细胞,阳性率在60%以上.结论:重组AAV携带的Nurrl基因能够在MSCs中表达,本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础.     

Nurr1基因在大鼠体外培养骨髓基质细胞的表达

目的 :为获得高效表达孤儿核受体 (orphannuclearreceptor,Nurr1)基因的骨髓基质细胞。 方法 :采用分子克隆技术 ,构建携带Nurr1基因的重组腺相关病毒 (adeno associatedvirus,AAV)载体 ;与包装质粒 pAAV RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK2 93制备具有感染活性的AAV Nurr1病毒粒子 ;用病毒上清液感染原代培养的大鼠骨髓基质细胞 (Marrowstromalcells,MSCs) ,并用免疫细胞化学方法检测阳性细胞。 结果 :经酶切鉴定和DNA测序 ,得到了序列正确的重组pAAV Nurr1;感染HT10 80细胞进行病毒滴度测定 ,每mL病毒贮存液可达 10 12 个阳性细胞 ;获得了Nurr1阳性的骨髓基质细胞 ,阳性率在 6 0 %以上。结论 :重组AAV携带的Nurr1基因能够在MSCs中表达 ,本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础。     

Nurrl基因在大鼠体外培养骨髓基质细胞的表达

目的：为获得高效表达孤儿核受体(orphan nuclear receptor，Nurrl)基因的骨髓基质细胞。方法：采用分子克隆技术，构建携带Nurrl基因的重组腺相关病毒(adeno-associated virus，AAV)载体；与包装质粒pAAV-RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK293制备具有感染活性的AAV-Nurrl病毒粒子；用病毒上清液感染原代培养的大鼠骨髓基质细胞(Marrow stromal cells，MSCs)，并用免疫细胞化学方法检测阳性细胞。结果：经酶切鉴定和DNA测序，得到了序列正确的重组pAAV-Nurr1；感染HTl080细胞进行病毒滴度测定，每mL病毒贮存液可达10^12个阳性细胞；获得了Nurrl阳性的骨髓基质细胞，阳性率在60％以上。结论：重组AAV携带的Nurrl基因能够在MSCs中表达，本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础。     

肿瘤血管抑制肽alphastatin重组腺伴随病毒的制备

目的构建并产生肿瘤血管抑制肽alphastatin(Al)重组腺伴随病毒(rAAV)载体。方法将目的基因alphastatin插入载体质粒pSSHG-巨细胞病毒(CMV)的EcoRI和BamHI位点,构建重组质粒pSSHG-CMV/NT4-Al。用腺病毒辅助质粒pFG140代替野生型腺病毒,包装质粒pAAV/Ad及已构建的重组腺伴随病毒载体质粒,使用三质粒共转染法转染293细胞,包装得到腺伴随病毒(AAV)-Al。采用氯仿抽提、聚乙二醇沉淀回收纯化,斑点杂交方法测定重组病毒滴度。结果重组病毒rAAV-Al滴度约为2×1012颗粒/ml。结论成功制备了重组病毒rAAV-Al,提供了一种生产足量安全的rAAV-Al简单易行的方法,为肿瘤的基因治疗实验奠定了基础。     

重组大鼠质粒pEGFP-GDNF的构建及真核细胞转染

目的　构建携带大鼠胶质细胞源性神经营养因子(GDNF)基因的真核细胞表达载体,为应用GDNF进行如帕金森综合征之类的神经元退化性疾病的基因治疗打基础。方法　采用RT- PCR方法从大鼠胎脑组织总RNA中扩增出该基因的c DNA序列,并克隆到增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体p EGFP- C1中,对重组质粒p EGFP- GDNF进一步鉴定。采用电转及阳离子脂质体将重组质粒p EGFP- GDNF转染至SH- SY5 Y细胞。结果　大鼠GDNF c DNA已正确地克隆到真核表达载体p EGFP- C1中,而构建成重组大鼠质粒p EGFP-GDNF。GDNF基因可稳定表达在细胞中。结论　真核细胞表达载体p EGFP- GDNF以及表达GDNF工程细胞SH-SY5 Y的成功构建,为进一步开展GDNF基因治疗PD等中枢神经系统疾病奠定了基础。     

脊髓源星形胶质细胞GFAP表达抑制真核表达载体构建及最佳短发夹样RNA分子筛选

目的构建并筛选大鼠胶质原纤维酸性蛋白（GFAP）表达抑制短发夹样RNA（shRNA）真核表达载体。方法针对GFAP基因全编码序列设计并合成三对9bp茎环结构、19bp干扰序列特异性shRNA模板，体外定向克隆构建特异性重组质粒真核表达载体；通过体外大鼠脊髓源星形胶质细胞GFAP表达抑制模型，脂质体介导RNA干扰分子转染，实时荧光定量RT—PCR及Wesem blot技术观察RNA干扰后原代星形胶质细胞GFAP表达抑制效果．筛选最佳GFAP表达干扰抑制真核表达载体。结果序列测定证实GFAP—shRNA重组质粒真核表达载体构建成功，三对shRNA模板在mRNA及蛋白表达水平抑制靶基因表达效率分别为81％、63％、56％。结论高效率的GFAP—shRNA真核表达载体在大鼠原代星形胶质细胞GFAP表达抑制模型中能高效抑制GFAP基因表达，为后续多靶点RNA干扰技术在脊髓损伤胶质瘢痕抑制基因治疗中的应用奠定了前期基础。     

人EGFL7基因短发夹结构RNA慢病毒载体的构建及鉴定

目的构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）慢病毒载体。方法将靶向EGFL7基因的短发夹结构RNA（shRNA）表达序列连接到包含U6启动子及绿色荧光蛋白（GFP）报告基因的慢病毒载体pGCL-GFP中,获得重组质粒,命名为pGCL-GFP-vshEGFL7,经多聚酶链反应（PCR）和测序鉴定后,与慢病毒包装质粒pHelper 1.0及pHelper 2.0通过lipofectamine 2000共转染至包装细胞293T,包装产生病毒液,测定其滴度。结果PCR扩增和测序结果证实EGFL7shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为4.8×107TU/ml。结论成功构建人EGFL7基因shRNA慢病毒载体。     

外源EGFP和hTH基因在恒河猴骨髓源神经干细胞内的稳定表达

目的构建携带人酪氨酸羟化酶(hTH)的荧光真核表达质粒-pEGFP-C2-hTH,转染骨髓基质细胞源神经干细胞(BMSCs-D-NSCs),观察外源EGFP和hTH基因在BMSCs-D-NSCs中的表达情况。方法应用基因重组技术,将pWAV2-TH中的TH目的基因亚克隆到荧光真核表达载体 pEGFP-C2,以酶切和测序鉴定重组质粒pEGFP-C2-hTH的正确性:pEGFP-C2-hTH经NucleofectorTM 核转染仪转染培养的恒河猴BMSCs-D-NSCs,24 h后观察绿色荧光蛋白的瞬时表达情况,10 d后行 TH单克隆抗体的免疫组化和TH基因的RT-PCR。结果 (1)酶切、PCR和DNA序列鉴定均证实插入片段的正确性;(2)细胞转染24 h后,荧光显微镜下可观察到绿色荧光蛋白(GFP)的表达,观察到 80％的转染细胞发出绿色荧光;转染10 d后细胞的RT-PCR检测到hTH基因的表达,TH单克隆抗体免疫组化结果显示转染细胞呈阳性染色,同时在荧光显微镜下观察到绿色荧光。结论构建的 hTH荧光真核表达重组质粒pEGFP-C2-hTH,经电转染方法转染至BMSCs-D-NSCs内,成功表达hTH 和EGFP,为BMSCs-D-NSCs基因治疗提供了实验基础。     

利用Helper-Free系统构建cdk5-siRNA腺相关病毒载体并鉴定

目的构建能表达CDK5特异性小干扰RNA(siRNA)(cdk5-siRNA)的重组腺相关病毒(AAV)载体,体外观察其对CDK5基因的沉默作用。方法采用基因克隆技术将合成的特异性cdk5 RNA干扰寡核苷酸序列克隆至AAV Helper-Free System中的表达质粒pAAV-MCS,构建出质粒pAAV-MCS-cdk5-siRNA,通过酶切测序鉴定重组质粒;将该质粒与系统中的控制质粒pAAV-RC、辅助质粒pHelper用磷酸钙法共转染HEK293细胞,包装得到表达cdk5-siRNA的重组腺相关病毒载体(rAAV-cdk5-siRNA);用斑点杂交法测定重组病毒的滴度,重组病毒感染体外培养的PC12细胞,W estern b lot检测其抑制cdk5表达的效果。结果成功构建并包装出重组腺相关病毒载体rAAV-cdk5-siRNA,病毒滴度达4×1013/m l,重组病毒感染后的PC12细胞cdk5表达明显下调。结论构建的重组腺相关病毒载体rAAV-cdk5-siRNA能明显干扰cdk5的表达,为将其进一步应用于神经变性疾病的治疗研究奠定了基础。     

重组腺相关病毒介导的Dystrophin小基因载体构建及在mdx鼠中表达

目的 :研究重组腺相关病毒载体 (rAAV)介导的人Dystrophin小基因 (SMCKA3 999)载体构建及在DMD模型鼠 (mdx鼠 )的表达。方法 :将SMCKA3 999质粒 ,包装质粒pXX2、腺病毒成分辅助质粒pXX6共转染 2 93细胞 ,包装重组腺相关病毒载体介导的SMCKA3 999基因 (rAAVSMCKA3 999) ,以斑点杂交法测定病毒滴度 ,将rAAVSMCKA3 999单点注射到mdx鼠腓肠肌 ,于注射后 7个月取肌肉提取蛋白质行Westernblot检测。结果 :经三质粒共转染法构建的rAAVSMCKA3 999病毒滴度为 5 0× 10 10 ,在mdx鼠骨骼肌表达持续 7个月以上。结论 :构建的rAAVSMCKA3 999载体为进一步DMD基因治疗研究奠定了基础。     

人SNCA及其致病突变基因的逆转录病毒pEGZ／MCS HA载体的构建

目的:克隆人野生型SNCA基因,构建野生型SNCA基因及其致病突变Ala30Pro、Ala53Thr的逆转录病毒表达载体。方法:通过逆转录聚合酶链式反应(RT-PCR)方法从人胎脑扩增SNCA基因,T-A克隆后测序。在此基础上利用单核苷酸差异引物定点诱变法构建其致病突变Ala30Pro、Ala53Thr的SNCA基因的逆转录病毒载体,并用这些重组逆转录病毒载体转染宿主细胞。结果:PCR、酶切及测序证明逆转录病毒表达载体构建成功。目的基因序列在宿主细胞成功表达。结论:人野生型SNCA基因及其Ala30Pro、Ala53Thr突变基因的重组逆转录病毒pEGZ/MCSHA载体的成功构建,为进一步构建表达野生型及Ala30Pro、Ala53Thr突变型SNCA的PD细胞模型奠定基础。     

骨髓间质干细胞作为基因载体治疗胶质瘤的研究进展

胶质瘤基因治疗的传统载体及神经干细胞都存在缺陷,近期研究表明骨髓间质干细胞作为胶质瘤治疗的新型载体,具有强大的迁移力、趋瘤性及低免疫原性和免疫调节功能等优点。     

Replication-Deficient Adenovirus Vector Transfer ofgfpReporter Gene into Supraoptic Nucleus and Subfornical Organ Neurons

The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 × 10⁶pfu) and sacrificed 1–7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successfulin vitrotransfection of cultured cells from the SON and SFO was also achieved with Ad (2 × 10⁶to 2 × 10⁸pfu). The expression of GFP inin vitrotransfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO–SON axis.     

Behavioral and Cellular Protection of Rat Dopaminergic Neurons by an Adenoviral Vector Encoding Glial Cell Line-Derived Neurotrophic Factor

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinson's disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.     

Vector therapy in family therapy

Summary Family psychiatry has evolved as a new system for the organization of psychiatric practice; it takes the family group rather than the individual as the functional unit.In therapy, as in all other aspects of family psychiatry, the family is the unit. Family Therapy includes all procedures for the treatment of the family and can be divided into Family Psychotherapy and Vector Therapy, the subject of this communication.A vector denotes a quantity which has direction. Force, including emotional force, is a quantity with direction, and therefore can be represented by a vector. Furthermore, as direction is a property of a vector, and direction implies movement, it results in a dynamic situation. The individual can be regarded as an element in the family's field of emotional forces, and both the individual and the family as elements in the community's field of emotional forces.Vector therapy is concerned with evaluating these forces and readjusting their patterning within the life space to produce a more harmonious field of emotional forces within and around the family.

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Zusammenfassung Familienpsychiatrie hat sich als ein neues Organisationssystem der psychiatrischen Praxis entwickelt. Sie nimmt als funktionale Einheit mehr die Familiengruppe als das Individuum.In der Therapie ist die Familie, wie in allen anderen Formen der Familienpsychiatrie, die Einheit. Familientherapie schließt alle Behandlungsverfahren für die Familie ein und kann in Familienpsychotherapie und Vektortherapie, dem Gegenstand dieser Mitteilung werden.Ein Vektor bedeutet eine Quantität mit einer bestimmten Richtung. Kraft, einschließlich emotionaler Kraft, ist eine Quantität mit einer Gerichtetheit und kann daher durch einen Vektor dargestellt werden. Da Gerichtetheit eine Eigenschaft eines Vektors ist und Bewegung einschließt, folgt weiterhin daraus eine dynamische Situation. Der Einzelne kann als ein Element im emotionalen Kräftefeld der Familie gesehen werden und beide — der Einzelne und damit die Familie — als Elemente im emotionalen Kräftefeld der Gemeinde.Vektortherapie befaßt sich mit der Bestimmung dieser Kräfte und ihrer Neuordnung innerhalb des Lebensraumes, um ein harmonischeres Feld der emotionalen Kräfte innerhalb der Familie und um sie herum herzustellen.

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Résumé La psychiatrie de famille est devenue un nouveau système dans l'organisation de la pratique psychiatrique; c'est le groupe familial plutôt que l'individu qui est considéré comme unité fonctionnelle.En thérapie comme dans tous les autres aspects de la psychiatrie de famille, la famille est l'unité. La thérapie de famille comprend tous les modes de traitement de la famille et peut être divisée en psychothérapie de famille et thérapie vectorielle, cette dernière étant le sujet de ce travail.Un vecteur indique une quantité et une direction. La force, y compris la force émotionnelle, est une quantité et une direction; elle peut par conséquent être représentée par un vecteur. De plus, la direction étant une propriété du vecteur et la direction impliquant un mouvement, il en résulte une situation dynamique. L'individu peut être considéré comme un élément dans le champ familial des forces émotionnelles, et l'individu et la famille tous deux comme des éléments dans le champ communautaire des forces émotionnelles. La thérapie vectorielle consiste à évaluer ces forces et à les réajuster afin de créer un champ de forces émotionnelles plus harmonieux au sein et autour de la famille.

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Based on paper given at the International Congress of Psychotherapy, Wiesbaden 1967.     

Vector Symbolic Architectures for Context-Free Grammars

Cognitive Computation - Vector symbolic architectures (VSA) are a viable approach for the hyperdimensional representation of symbolic data, such as documents, syntactic structures, or semantic...     

A Twin Multi-Class Classification Support Vector Machine

Twin support vector machine (TSVM) is a novel machine learning algorithm, which aims at finding two nonparallel planes for each class. In order to do so, one needs to resolve a pair of smaller-sized quadratic programming problems rather than a single large one. Classical TSVM is proposed for the binary classification problem. However, multi-class classification problem is often met in our real world. For this problem, a new multi-class classification algorithm, called Twin-KSVC, is proposed in this paper. It takes the advantages of both TSVM and K-SVCR (support vector classification-regression machine for k-class classification) and evaluates all the training points into a “1-versus-1-versus-rest” structure, so it generates ternary outputs { ?1, 0, +1}. As all the samples are utilized in constructing the classification hyper-plane, our proposed algorithm yields higher classification accuracy in comparison with other two algorithms. Experimental results on eleven benchmark datasets demonstrate the feasibility and validity of our proposed algorithm.