MiRNA-132上调脑源性神经营养因子表达抑制β-淀粉样蛋白诱导的神经元损伤研究

研究背景脑源性神经营养因子(BDNF)在阿尔茨海默病(AD)发病机制中发挥重要作用,微小RNA-132(mi RNA-132)在神经元呈高表达,可以通过调控靶基因表达参与BDNF介导的神经发育过程。本研究旨在探讨阿尔茨海默病神经元模型中mi RNA-132与BDNF的调控关系和神经保护作用。方法体外培养海马神经元72 h后慢病毒转染mi RNA-132,并于体外培养第7天以β-淀粉样蛋白(Aβ)处理制备阿尔茨海默病神经元模型;实时荧光定量聚合酶链反应观察对照组与AD组mi RNA-132表达差异以及不同处理组BDNF m RNA表达变化,噻唑蓝法观察不同处理方式对细胞活性的影响。结果 (1)AD组海马神经元mi RNA-132(t=13.888,P=0.000)和BDNF m RNA(t=-12.274,P=0.000)表达水平均低于对照组。(2)原代培养的海马神经元经慢病毒转染后倒置相差荧光显微镜可见绿色荧光蛋白,对照组(t=16.135,P=0.000)和AD组(t=8.656,P=0.000)转染过表达mi RNA-132后均能上调BDNFm RNA表达。(3)AD组海马神经元活性降低(t=-6.023,P=0.000),AD组转染mi RNA-132后神经元活性增强(t=3.385,P=0.007),予以外源性BDNF共培养后神经元活性明显改善(t=3.672,P=0.004)。结论阿尔茨海默病神经元模型mi RNA-132和BDNF表达水平均下降,mi RNA-132可上调BDNF表达,提示mi RNA-132和BDNF对阿尔茨海默病神经元模型具有神经保护作用,有望为阿尔茨海默病诊断与治疗提供新的视角。     

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。     

再程序化脂肪源性干细胞的制备及体外定向分化为神经元的实验研究

目的制备再程序化脂肪源性干细胞以及探索诱导其定向分化为神经元的方法。方法体外培养脂肪来源干细胞(ADSCs),取纯化、鉴定过的第3代ADSCs接种于24孔板中并分A,B,C三组:A组为带有绿色荧光基因(GFP)的慢病毒载体介导Neurogenin2(Ngn2)基因转染的ADSCs,制备再程序化脂肪干细胞;B组为带有GFP的空载体病毒转染的ADSCs;C组为未进行慢病毒介导基因转染的ADSCs;转基因7d后加入含细胞生长因子诱导培养基诱导分化15d。光镜下观察各组细胞形态变化以及免疫荧光研究各组ADSCs诱导后定向分化为神经元的差异。结果与B组和C组相比,A组ADSCs转基因后再经诱导分化15d后绝大部分细胞类似神经元,胞体呈梭形或椭圆形,有两个或三个突起伸出,细胞表达神经丝蛋白NF、神经元特异性蛋白NeuN及神经元特异性烯醇化酯酶NSE比例大大提高。B组与C组的神经元分化效率,无显著差异,P>0.05。结论慢病毒介导Neurogenin2基因体外转染ADSCs可以制备出再程序化脂肪源性干细胞,诱导后具有更强的定向分化为神经元的能力。     

绿色荧光蛋白基因腺病毒载体转染人骨髓间充质干细胞的实验研究

目的研究绿色荧光蛋白基因（Ad—GFP）腺病毒载体在人骨髓间充质干细胞（BMSCs）转染和表达的量效关系及对细胞生物学特性的影响．探讨用该载体构建基因修饰BMSCs的可行性。方法在体外分离培养人BMSCs，流式细胞仪检测细胞免疫表型，293细胞包装制备病毒，以不同滴度的Ad—GFP（1×10^3-1×10^10PFU／mL）转染BMSCs．细胞计数法分析转染率．倒置显微镜观察细胞形态学改变．CCK8法检测细胞增殖活性，用血清撤离加入β-巯基乙醇诱导转染Ad—GFP的BMSCs向神经元样细胞定向分化。结果3～6代BMSCs表面标志CD34、CD45呈阴性而CD29、CD44呈阳性：当病毒滴度为1×10^7PFU／mL时转染率为55％，1×10^9及1×10^10PFU／mL滴度时转染率均为85％，但1×10^10PFU／mL滴度时出现细胞病理现象；7d荧光表达最强，28d仍可见荧光表达。荧光显微镜下可见表达Ad—GFP的BMSCs有三种亚群结构：滴度≥1×10^6PFU／mL时，BMSCs增殖在早期受到抑制，且呈剂量依赖；转染Ad—GFP的BMSCs经β一巯基乙醇诱导可分化为神经元样细胞，神经元特异性烯醇化酶（NSE）阳性。结论合适滴度的Ad—GFP可以高效转染BMSCs，对细胞的生物学特性影响较小，不影响诱导分化功能，BMSCs可以作为用Ad—GFP载体系统进行基因治疗研究的种子细胞。     

Ad-GFP转染兔离体基底动脉环的研究

目的 通过将将携带有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）转染兔离体基底动脉环,观察GFP表达部位及表达量的变化。方法 获取兔基底动脉环,置入含DMEM的96孔板中;加入1.8×106 pfu/μl的Ad-GFP转染1 h;转染后即刻3、5、7 d行冰冻切片,荧光显微镜观察亮绿色物质。另外,将不同病毒浓度（分别为1.2×105、1.2×106 、1.8×106和2.4×106 pfu/μl）转染离体血管环1 h后,培养3 d后行冰冻切片,用荧光显微镜观察结果。结果 荧光显微镜下观察发现,基底动脉环管壁各层均表达GFP,但主要位于血管内皮层,1 000倍下在内皮细胞内中明确观察到荧光颗粒。1.8×106 pfu/μl的Ad-GFP转染后发现,GFP在转染3 d后表达最明显,5 d次之,而7 d后表达最弱;不同浓度Ad-GFP转染3 d后发现,浓度为1.2×105 pfu/μl时,管壁有较弱绿色荧光,浓度不断升高亮度有所增强,浓度为1.8×106 pfu/μl时达到最高峰,浓度继续增加时荧光亮度未见进一步增强。结论 Ad-GFP可以成功转染入离体血管环中,以内皮层为主;3 d为病毒转染后最佳培养时间。     

bFGF荧光真核表达载体在骨髓间充质干细胞中的表达

目的探讨碱性成纤维细胞生长因（bFGF）荧光真核表达-载体转染骨髓间充质干细胞（MSCs）的毒达情况，为进一步开展bFGF基因治疗中枢神经系统疾病奠定实验基础。方法应用脂质体介导的基因转移技术将bFGF真核表达-载体导入MSCs，并于转染后18h在倒置荧光显微镜下观察绿色荧光蛋白的表达情况。结果与结论经转染后18h的MSCs在倒置荧光显微镜下可见片状荧光，说明转染成功。MSCs有望成为bFGF基因治疗中枢神经系统疾病的理想载体。     

转染绿色荧光蛋白的神经干细胞移植于半横断脊髓损伤处的体内外分化研究

目的 观察转染绿色荧光蛋白(GFP)的大鼠脊髓神经干细胞移植于半横断脊髓损伤处的体内外分化情况.方法 将表达GFP的慢病毒载体转染胎鼠脊髓神经干细胞,体外用10%胎牛血清诱导分化.转染后的神经干细胞与PLGA支架移植于大鼠半横断脊髓损伤处,术后1个月和3个月取材,行GFAP、NF和CNP免疫荧光染色.结果 转染GFP的神经干细胞球表达强烈的绿色荧光,体外分化可见GFAP/GFP、NF/GFP和CNP/GFP双阳性细胞,GFAP/GFP双阳性细胞明显多于其他两种.移植后3个月,GFP阳性细胞在脊髓内明显减少,可见少数GFAP/GFP和CNP/GFP舣阳性细胞,未见NF/GFP双阳性细胞.结论 转染GFP的神经干细胞可在体外增殖和分化,但大部分分化成胶质细胞.移植于急性期脊髓损伤处的神经干细胞不被诱导分化成神经元样细胞,可被诱导分化成神经胶质细胞.     

大鼠三叉神经脊束核尾侧亚核浅层内GABA样、L—ENK样，GlyT2样阳性末梢与Sindibis病毒转染表达GFP神经元之间的联系

应用GFP基因重组病毒标记技术和免疫荧光组织化学技术 ,在荧光显微镜和激光共聚焦扫描显微镜下观察了大鼠三叉神经脊束核尾侧亚核 (Vc)浅层内GABA样、L ENK样、GlyT2样阳性纤维和末梢与Sindibis病毒转染表达GFP神经元之间的联系。结果显示 :①将GFP基因重组Sindibis病毒注入一侧Vc浅层后 ,仅在同侧Vc注射部位附近的Ⅰ～Ⅲ层内观察到 4～ 8个逆标的GFP神经元 ,这些神经元的胞体为小型 (直径≤ 15 μm)圆形、梭形或不规则形 ;②Vc浅层内均可见大量的GABA样、L ENK样、GlyT2样阳性纤维和末梢 ,以Ⅰ和Ⅱ层分布最为密集 ;③GABA样、L ENK样、GlyT2样阳性纤维和末梢分别聚集于GFP标记神经元的胞体或树突周围 ,并与之形成密切接触。以上结果表明 :Vc浅层内GFP神经元可能与GABA能、L ENK能、Gly能阳性纤维和末梢形成突触联系并接受这些抑制性神经末梢的调控。     

大鼠三叉神经脊束核尾侧亚核浅层内GABA样、L-ENK样、GlyT2样阳性末梢与Sindibis病毒转染表达GFP神经元之间的联系

应用GFP基因重组病毒标记技术和免疫荧光组织化学技术,在荧光显微镜和激光共聚焦扫描显微镜下观察了大鼠三叉神经脊束核尾侧亚核(Vc)浅层内GABA样、L-ENK样、GlyT2样阳性纤维和末梢与Sindibis病毒转染表达GFP神经元之间的联系.结果显示:①将GFP基因重组Sindibis病毒注入一侧Vc浅层后,仅在同侧vc注射部位附近的Ⅰ～Ⅲ层内观察到4～8个逆标的GFP神经元,这些神经元的胞体为小型(直径≤15 μm)圆形、梭形或不规则形;②Vc浅层内均可见大量的GABA样、L-ENK样、GlyT2样阳性纤维和末梢,以Ⅰ和Ⅱ层分布最为密集;③GABA样、L-ENK样、GlyT2样阳性纤维和末梢分别聚集于GFP标记神经元的胞体或树突周围,并与之形成密切接触.以上结果表明:Vc浅层内GFP神经元可能与GABA能、L-ENK能、Gly能阳性纤维和末梢形成突触联系并接受这些抑制性神经末梢的调控.     

慢病毒转染胶质细胞源性神经营养因子脂肪干细胞脑移植的研究

目的 探讨慢病毒转染胶质细胞源性神经营养因子(GDNF)的脂肪干细胞(ADSCs)脑移植的应用价值.方法 分离、培养SD大鼠ADSCs,用高滴度慢病毒将GDNF转染入细胞,观察细胞形态及用免疫荧光法观察神经标志物的表达.用立体定向仪将转染ADSCs植入大鼠的纹状体,移植1个月和2个月后,用荧光显微镜观察ADSCs脑内存活和分布状况,用Western blot检测GDNF蛋白的表达,并与正常对照组比较.结果 慢病毒转染ADSCs形态类似神经元,胞浆神经元特异性烯醇化酶(NSE)表达阳性.ADSCs移植到大鼠纹状体1个月后,移植部位有大量存活的ADSCs,2个月后,仍有部分存活细胞,并向远处移行,GDNF蛋白水平显著高于正常对照组(均P<0.01).结论 慢病毒转染GDNF的ADSCs可为干细胞移植治疗中枢神经系统疾病提供新的供体.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

帕金森病运动症状进展分析

目的分析帕金森病(PD)患者运动症状进展特点。方法采用PD统一评分量表(UPDRS)Ⅲ对912例PD患者进行评估。结果与病程1年的患者比较,除病程1~2年的患者外,其他病程患者的UPDRSⅢ评分、强直分、姿势或步态异常分、轴性症状总分、言语分、步态分显著升高(均P0.05),病程5~6年及14年患者的震颤分,病程5~6年、7~8年、9~13年、14年患者的运动迟缓分、姿势分显著升高(P0.05~0.01)。轴性症状进展速度高于UPDRSⅢ评分。结论 PD患者病程早期UPDRSⅢ评分进展快,震颤症状进展独立于其他症状,轴性症状评分较UPDRSⅢ更敏感地反映疾病加重趋势。     

Frequency of accumulation of filaments in adaxonal cytoplasm of Schwann cells of myelinated fibers of rat spinal roots

Summary The frequency of accumulation of 6-nm filaments in the adaxonal cytoplasm of Schwann cells in the 6th lumbar dorsal and ventral roots was evaluated in 4-, 8-, 26- and 45-week-old Sprague-Dawley rats. The frequency was higher in 4- and 8-week-old (growing) rats than in 26- and 45-week old (mature) rats, and also higher in ventral than in dorsal roots in 4-, 8- and 26-week old rats. There were no clusters on certain groups of myelinated fibers according to the size of transverse axonal area, in both the ventral and dorsal roots. Therefore, this accumulation may reflect certain functions of the adaxonal cytoplasm of Schwann cell during natural growth and maturation of the axon and myelin sheath.     

Function of circle of Willis

Nearly 400 years ago, Thomas Willis described the arterial ring at the base of the brain (the circle of Willis, CW) and recognized it as a compensatory system in the case of arterial occlusion. This theory is still accepted. We present several arguments that via negativa should discard the compensatory theory. (1) Current theory is anthropocentric; it ignores other species and their analog structures. (2) Arterial pathologies are diseases of old age, appearing after gene propagation. (3) According to the current theory, evolution has foresight. (4) Its commonness among animals indicates that it is probably a convergent evolutionary structure. (5) It was observed that communicating arteries are too small for effective blood flow, and (6) missing or hypoplastic in the majority of the population. We infer that CW, under physiologic conditions, serves as a passive pressure dissipating system; without considerable blood flow, pressure is transferred from the high to low pressure end, the latter being another arterial component of CW. Pressure gradient exists because pulse wave and blood flow arrive into the skull through different cerebral arteries asynchronously, due to arterial tree asymmetry. Therefore, CW and its communicating arteries protect cerebral artery and blood–brain barrier from hemodynamic stress.     

Effects of prevention of afferentation of the development of the chick optic lobe

BONDY, S. C., M. E. HARRINGTON AND C. L. ANDERSON. Effects of prevention of afferentation on the developmentof the chick optic lobe. BRAIN RES. BULL. 3(5) 411–413, 1978.—The effects of unilateral extirpation of the right optic cup of the three-day incubated chick embryo upon the rate of synthesis and the stability of DNA in the non-innervated optic lobe, have been studied. This surgical procedure prevents innervation of the optic lobe contralateral to the removed eye, while the other optic lobe is normally innervated by retinal ganglion cells of the remaining eye. At the 20th day of incubation, the DNA content of the non-innervated lobe was below that of the paired lobe receiving normal innervation. This deficiency of cell number was caused by two events; death of an excess number of neurons formed early in embryogenesis and a reduced rate of glial proliferation in the later stages of incubation.     

Modelling of movement of the lamprey: Control of oscillators

This article discusses the control methods of the central pattern generator (CPG). First a control model of the CPG is presented using 2 oscillators, and we suggest that phasic modulation to the CPG by means of phasic information is effective for controlling the phase difference between oscillators. Next, two models for controlling the CPG of a lamprey are proposed. One model describes a control system from the brain stem, in which the reticulospinal neurons control the CPG by receiving feedback signals and sending control signals to the neck region of the CPG. The other is a model for learning an localized control system to generate a desired motor pattern. By means of these models, a role of the efference copy is suggested.     

利培酮对精神分裂症患者生活质量的影响

目的：比较利培酮与氟哌啶醇对精神分裂症患者生活质量的影响。方法：对门诊72例服用氟哌啶醇及74例服用利培酮的精神分裂症患者用生活质量综合评定问卷（GQOLI）、阳性与阴性症状量表（PANSS）、副反应量表（TESS）进行评定。结果：利培酮组患者治疗后生活质量有所提高,而氟哌啶醇组患者生活质量有所下降。结论：利培酮治疗有利于患者提高生活质量。     

腺垂体功能减退症临床特征变化分析

目的探讨腺垂体功能减退症患者的病因结构变化及临床表现。方法回顾性分析我院2013-01—2016-12住院及门诊78例腺垂体功能减退症患者的临床资料。结果男32例(41.03%),女46例(58.97%);诊断时年龄11~89岁,平均62.5岁;鞍区占位(包括术前及术后)52例(66.67%),席汉综合征8例(10.26%),空泡蝶鞍9例(11.65%),病因不明8例(10.26%),垂体-下丘脑发育不良1例(1.28%)。首次就诊科室:纳差厌食、恶心呕吐就诊于消化内科36例(46.15%)最常见。ACTH+TSH+Gn+G激素缺乏为19例最多,占24.36%,ACTH+TSH+Gn缺乏15例,占19.23%。结论腺垂体功能减退症病因结构发生变化,发病人群、首发症状及受累激素也不同,患者女性多于男性,发病年龄偏高,症状不典型,分布于临床多个科室,其中以低钠血症为首发临床表现就诊消化内科最多。     

Standards of instrumentation of EMG

Standardization of Electromyography (EMG) instrumentation is of particular importance to ensure high quality recordings. This consensus report on “Standards of Instrumentation of EMG” is an update and extension of the earlier IFCN Guidelines published in 1999. First, a panel of experts in different fields from different geographical distributions was invited to submit a section on their particular interest and expertise. Then, the merged document was circulated for comments and edits until a consensus emerged.The first sections in this document cover technical aspects such as instrumentation, EMG hardware and software including amplifiers and filters, digital signal analysis and instrumentation settings. Other sections cover the topics such as temporary storage, trigger and delay line, averaging, electrode types, stimulation techniques for optimal and standardised EMG examinations, and the artefacts electromyographers may face and safety rules they should follow. Finally, storage of data and databases, report generators and external communication are summarized.     

《绵阳市社会心理服务工作管理办法》解读

2018年,国家卫生健康委员会等10部委联合发布《关于印发全国社会心理服务体系建设试点工作方案的通知》,四川省绵阳市被列为全国第一批试点地区。绵阳市人民政府依据《中华人民共和国精神卫生法》等相关法律法规和文件精神,结合前期调查研究和社会心理服务工作的试点实际,编制出台了《绵阳市社会心理服务工作管理办法》,并于2021年12月25日起施行。本文围绕社会心理服务的相关概念、办法总则、重点内容、保障措施等方面进行解读,以期为社会心理服务工作的规范、持续和有效开展提供参考。