体外培养人脑动静脉畸形血管内皮细胞中VEGF表达增加

目的探讨体外培养的人脑动静脉畸形血管内皮细胞和正常脑血管内皮细胞中血管内皮细胞生长因子(VEGF)表达水平的差异。方法采用组织块贴壁法对人脑动静脉畸形血管内皮细胞进行培养和形态学观察;免疫组化检测CD31和vWF抗原验证内皮细胞;采用RT-PCR和Western blot技术检测脑动静脉畸形血管内皮细胞VEGF mRNA和蛋白表达,并与和正常脑血管内皮细胞进行比较。结果体外培养的内皮细胞CD31和vWF染色阳性率达95%以上。人脑动静脉畸形血管内皮细胞中VEGF mRNA和蛋白表达均显著升高(P0.05)。结论体外培养的人脑动静脉畸形内皮细胞中VEGF的高表达,提示血管生成在脑动静脉畸形的发病机制中起重要作用。     

人脑动静脉畸形血管内皮细胞的体外培养和VEGF、Tie受体及血管生成素表达

目的为获取较纯净的脑动静脉畸形(AVM)血管内皮细胞，以探讨血管内皮细胞生长因子(VEGF)、Tie受体及血管生成素(Ang)在脑AVM病变进展中的作用。方法组织块贴壁法对AVM血管内皮细胞进行培养和形态学观察。采用免疫组化(ABC法)检测内皮细胞第八因子相关抗原(FⅧ-RA)、VEGF、Tie1、Tie2、Ang1、Antg2阳性表达。结果相差显微镜下，培养的活细胞具有单层“卵石样”排列的典型特征，FⅧ-RA免疫阳性表达率超过95％，证实为血管内皮细胞。内皮细胞中VEGF、Tie2和Ang2表达显著，而Tie1及Ang1的表达较弱。结论脑AVM内皮细胞可以获取和培养繁殖，可供研究血管新生和脑血管畸形发病机理的体外模型；VEGF、Tie受体及Ang可能与脑AVM的形成、扩展和破裂出血有关。     

缺氧诱导人脑动静脉畸形血管内皮细胞生长因子表达

目的 探讨缺氧条件下体外培养的人脑动静脉畸形（cAVM）血管内皮细胞中血管内皮细胞生长因子（VEGF）基因表达与细胞超微结构变化。方法 采用组织块贴壁法对cAVM血管内皮细胞进行培养和形态学观察。采用免疫组化方法检测细胞中第八因子相关抗原（FⅧ-RA）的表达，采用RT-PCR技术观察静态和缺氧状态下2h、4h、8h血管内皮细胞VEGF mRNA表达。采用酶联免疫吸附实验（ELISA）检测每组细胞上清液中VEGF蛋白含量；在透射电镜下观察细胞超微结构的变化。结果 体外培养的活细胞具有单层“卵石样”排列的典型特征．95％以上的细胞FⅧ-RA染色阳性。血管内皮细胞vEGF蛋白和mRNA表达在缺氧2h开始升高（P〈0．05），并持续至缺氧8h（P〈0．01）：细胞线粒体丰富。核糖体及粗面内质网逐渐增多。结论 缺氧可能在基因转录水平诱导VEGF表达，后者通过自分泌途径刺激cAVM血管内皮细胞增殖。     

脑动静脉畸形血管内皮细胞和平滑肌细胞的体外培养

目的：探讨脑动静脉畸形（AVM）内皮细胞和平滑肌细胞的分离、培养及鉴定方法。方法：手术获得脑AVM标本后,分别采用组织块贴壁法和酶消化法对脑AVM血管内皮细胞、平滑肌细胞进行培养和形态学观察,采用免疫组化分别检测培养的内皮细胞CD31抗原和平滑肌细胞SMA抗原阳性表达。结果：相差显微镜下,培养的内皮细胞呈扁平梭形,胞核椭圆居中;平滑肌细胞呈长梭形。CD31和SMA分别在两种细胞中免疫阳性表达率超过90％。结论：AVM内皮细胞、平滑肌细胞可以获取和培养增殖,为可供研究脑血管畸形血管生物学的体外模型。     

人外周血内皮祖细胞培养方法的建立

背景：成人外周血来源丰富,但内皮祖细胞含量较少,为使其能够更好的应用于组织工程及细胞治疗,有必要建立外周血内皮祖细胞成熟、稳定的体外扩增体系。 目的：建立稳定的人外周血分离、培养和体外扩增血管内皮祖细胞的方法。 方法：应用密度梯度离心法,获取外周血单个核细胞,将分选后细胞接种于预先包埋了人纤维连接蛋白的培养板上,加入内皮祖细胞专用培养基中培养3 d后,洗掉非贴壁细胞,培养至第6天,收集贴壁细胞,应用倒置显微镜和苏木精-伊红染色进行细胞形态学观察;采用MTT法和细胞计数测定第1,3代细胞生长曲线;应用流式细胞仪测定祖细胞和内皮细胞系标志,对培养的细胞进行鉴定。 结果与结论：细胞生长曲线测定表明接种后第3天细胞进入指数增生期,至第6天进入平台期,随着传代次数的增加,细胞增殖速度变慢,同时表达干细胞表面标志CD34、CD133和内皮细胞表面标志血管性血友病因子、血管内皮生长因子受体2。证明人外周血可以分离培养内皮祖细胞。     

大鼠骨髓基质干细胞体外分离、培养及分化的相关实验研究

目的探索大鼠骨髓基质干细胞(BMSC)体外分离、培养及纯化的合适实验条件,并探讨其在体外定向诱导分化为神经元样细胞的可行性。方法通过密度梯度离心法从成年大鼠骨髓中分离出BMSC,而后通过贴壁培养法培养及纯化,观察其生长特性;对纯化后的BMSC使用碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)进行定向诱导分化,并进行免疫细胞化学鉴定。结果体外培养的BMSC传4代后,纯度最高,可达(95.23±3.06)%;其诱导分化7 d后,(75.43±7.63)%的细胞表现为-βTubulinⅢ阳性的神经元样细胞,(33.01±6.73)%的细胞则为GFAP阳性的胶质细胞。结论BMSC在体外培养条件下生长良好,经bFGF和EGF诱导后可大量分化为神经元样细胞和神经胶质细胞。     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

大鼠脑微血管内皮细胞的体外培养

目的探讨大鼠脑微血管内皮细胞的培养方法.方法取Wistar大鼠乳鼠脑组织,采用筛网过滤、胶原酶消化、离心等技术获取脑微血管内皮细胞,并进行培养.通过形态学、免疫细胞化学方法进一步鉴定,采用MTT方法测定生长曲线.结果经形态学、免疫细胞化学方法鉴定所培养的细胞为脑微血管内皮细胞,观察到原代脑微血管内皮细胞有3种表型,细胞呈单层生长,并可传代培养.结论建立大鼠脑微血管内皮细胞的培养方法可为体外研究脑血管病提供有益帮助.     

不同种属骨髓间充质干细胞体外培养特性的比较研究

目的比较体外培养条件下不同种属骨髓间充质干细胞(BMSC)的生物学特性。方法采用贴壁法和离心法,对小鼠、大鼠、家兔和人BMSC进行分离培养。采用光镜和电镜观察细胞形态,MTT法测定生长曲线,免疫荧光细胞化学法和流式细胞仪分析表面分子Stro-1的表达,并通过试剂盒检测碱性磷酸酶(ALP)活性、免疫组化检测骨钙素表达以及Von Kossa法显示钙盐沉积等手段评价BMSC对成骨诱导液(10 nmol/L地塞米松,10 mmol/L磷酸甘油和50 mg/L抗坏血酸)的反应性。结果贴壁法细胞得率是离心法的10余倍。各种属BMSC光镜下形态不同,电镜下形态相似;生长曲线基本一致。人原代贴壁细胞Stro-1阳性率为(91.4±8.3)%,小鼠为(83.5±6.2)%。在成骨诱导液中,小鼠BMSC向脂肪细胞分化,家兔BMSC死亡,大鼠和人BMSC可被成功诱导为成骨细胞。BMSC在培养中有自发分化现象。结论小鼠、大鼠、家兔和人BMSC可在体外被大量扩增,所得到的细胞是以低(未)分化细胞为主、不同分化程度的细胞混合物。不同种属BMSC在形态学和对同一诱导液的反应性上存在差异。     

神经干细胞条件培养液对脊髓神经组织细胞生长的作用

目的 探讨不同浓度神经干细胞条件培养液对体外培养脊髓组织中不同细胞成分生长的影响.方法 从胚胎脑组织中分离、培养神经干细胞;从胚胎脊髓中分离脊髓神经组织成分.采用免疫组织化学方法对分离培养的神经干细胞及培养的脊髓神经细胞进行染色鉴定.将脊髓神经组织细胞悬液置于不同浓度的神经干细胞条件培养液中培养.分析不同浓度的干细胞条件培养液对不同细胞成分生长的影响.结果 与对照组比较,30%及50%浓度的神经干细胞条件培养液明显促进人脊髓神经元的生长,而其它浓度条件液对胶质细胞生长作用较强.结论 人胚胎神经干细胞条件培养液对体外培养的脊髓神经元或胶质细胞促生长作用的差异与其浓度有关.     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

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     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.     

Summary of Legislation of 1933 of interest to the Department of Mental Hygiene

Psychiatric Quarterly -