卒中后吞咽障碍的分子生物学研究进展

卒中后吞咽障碍是卒中后常见并发症,指患者由于卒中导致吞咽过程中的困难表现,其发生、发展的分子机制尚未阐明.调控吞咽功能的两个关键区域孤束核和疑核,其分泌的多种神经递质与吞咽功能密切相关.与孤束核密切相关的5-羟色胺(5-hydroxytryptamine,5-HT)及其受体5-HT1A、谷氨酸及N-甲基-D-天冬氨酸受...     

功能磁共振成像在脑梗死患者运动功能评价中的作用探讨

目的 利用功能磁共振成像（functional magnetic resonance imaging,fMRI）分析单侧放射冠梗死患者大脑半球的激活部位及偏侧化指数（laterality index,LI）与运动功能康复水平的关系。方法 选取12例单侧放射冠梗死的患者为卒中组,5例正常志愿者为对照组。所有入选者均进行血氧水平依赖功能磁共振成像（blood oxygenation level dependent-functional magnetic resonance imaging,BOLD-fMRI）检查,扫描设备为德国西门子3.0T磁共振扫描系统。试验采用Block设计,采取患手顺序对指任务。扫描结果采用统计参数图（Statistical Parametric Mapping,SPM2）进行数据分析和脑功能区定位,计算不同感兴趣区激活体素数目及LI。扫描结束后记录患者上肢运动功能评分（Fugl-Meyer评分,F-M评分）,分析LI与F-M评分之间的相关性。结果 与对照组相比,卒中组脑部激活范围较广泛,表现为双侧运动传导通路的激活;双侧大脑半球、初级感觉运动区（sensory motor cortex,SMC）、第一躯体运动区（M1区）LI明显减少（P=0.004,0.008,0.027）。卒中组LI（半球、SMC、M1）与F-M评分之间的相关性无统计学意义（r=0.133,P=0.618;r=0.558,P=0.059;r=0.297,P=0.348）;卒中组最强激活点位于中央前回（Precentral gyrus,PRE）患者F-M评分较高（52±22）,最强激活点位于中央后回（postcentral gyrus,POS）患者F-M评分较低（36±27）,以上两组F-M评分之间差异无统计学意义（P>0.05）。结论 fMRI可以显示卒中患者运动康复过程中功能区的变化;单侧放射冠脑梗死后,与运动任务相关的脑区激活范围存在明显偏侧化现象;偏侧化程度与患者上肢运动功能之间可能无明显关系。     

Basic Concepts of Molecular Biology for the Epileptologist

Summary: Fundamental techniques used in molecular biology can be applied toward questions of relevance to epilepsy. Many of the most common techniques used for working with nucleic acids, including DNA extraction, electrophoresis, cloning in plasmid vectors, making probes, and the polymerase chain reaction are now commonly used in basic epilepsy research. Some specific approaches that can be used to address particular questions are methods for identifying a human gene (Southern analysis and screening a library), determining whether a gene is expressed in a given brain region (Northern analysis and in situ hybridization), and expressing a gene in tissue culture or a whole organism (cell transfection and transgenic animals).     

Detection of Neurofibrillary Tangles by the Methenamine-Silver Method: Comparison with Other Silver Stains and with Immunostaining Methods

Recently we devised the methenamine-silver (M-S) staining method for detecting amyloid of senile plaques. Subsequently it was noticed that this procedure also readily stains neurofibrillary tangles (NFT) and neuropil threads. However, the sensitivity of the M-S method as an NFT stain has not been determined. In order to clarify this point, we performed a quantitative study on NFT, comparing the M-S method with conventional Bodian method, Gallyas-Braak method, and Bielschowsky-Hirano method on brain samples from three Alzheimer-type dementia patients and two non demented subjects. In addition, NFT staining by the M-S method was also compared with staining by anti-tau and anti-ubiquitin antibodies. Our results indicate that the M-S method was as sensitive for NFT as the Gallyas-Braak procedure. Further-more, the M-S method stained not only intraneuronal NFT, but also extracellular NFT, some of which could not be identified by conventional silver staining techniques and by immunostaining for tau and ubiquitin. We conclude that the M-S method is useful for the routine examination of senile plaques and NFT.     

假肥大型肌营养不良症患者HLA-A、B、DR基因表达研究

目的 研究南方假肥大型肌营养不良症(duchenne muscular dustrophy，DMD)患者HLA-A、B、DR基因多态性，探讨免疫遗传因素在DMD发病中的作用，并为临床骨髓移植提供基础性资料。方法 采用PCR反向序列特异性寡核苷酸杂交技术与美国骨髓库编码软件(National marrow donor program，NMDP)，对29例DMD患者HLA-A、B、DR等位基因多态性进行研究。结果 DMD组HLA-A24等位基因频率9.03％，与对照组22.16％相比有所降低(P=0.017)；DMD组HLA-B13等位基因频率16.95％，与对照组6.75%相比显著增高(P=0.007)。但校正后P值差异均无显著性(P值分别为0.255和0.231)。结论 DMD患者HLA-A、B、DR基因表达与正常对照组差异无显著性，HLA遗传易感性与DMD发病无明显相关，但尚需要扩大样本量或进行高分辨加深研究。     

Molecular Biology of 'Szheimer's Disease and Neuron's Death

'Szheimer's disease has aroused much interest among neuroscientists within the recent decades. The deposition of amyloid β-protein in senile plaques is one of the major h'slmarks of the disease, and is thought to be a primary factor for its occurrence. Recent studies have shown that amyloid β-protein causes degeneration of cultured hippocamp's neurons, and that its neurotoxicity is enhanced by its aggregation and by conformation's changes. Therefore, factors that stimulate or inhibit amyloid β-protein aggregation may play key roles in the etiology of 'Szheimer's disease. Moreover, environment's factors, such as 'suminum and zinc, as well as genetic factors may 'sso be involved in the pathogenesis of the disease.     

Heterogeneity Among Neuroepithelial Cells in the Chick Retina Revealed by Immunostaining with Monoclonal Antibody PM1

Neuroepithelial cells appear as a homogeneous population of cells in the cell cycle that seem to behave as pluripotent neural precursors. The study of the intrinsic heterogeneity and subtle developmental changes among neuroepithelial cells has been hindered by the lack of specific markers. To address that study, a panel of monoclonal antibodies was produced against early developing chick retina. The monoclonal antibody precursor marker 1 (PM1) labels most, if not all, of the early neuroepithelial cells in embryonic day 4 retinal sections. This pattern is transient since the labelling becomes restricted to the peripheral retina as development proceeds and eventually disappears from the neuroepithelial cells. However, apparently in parallel, the differentiating retinal ganglion cells become PM1-positive. The expression of the PM1 antigen, a 73 × 10³ M _r protein, as shown by western blotting, also decreases with development. In addition, a chick retina dissociated-cell culture system, where retinal neuroepithelial cells actively proliferate and undergo differentiation under defined conditions, in combination with monoclonal antibody PM1, allowed us to characterize and quantify the proliferating and differentiating neuroepithelial cells. Interestingly, the fraction of total neuroepithelial cells that are stained with PM1 sharply decreases as retinal development proceeds, in correlation with the staining pattern in sections from matched stages. These data thus reveal that the pluripotent neural precursors in the chick retina already represent an intrinsically heterogeneous population, and that this population changes with development.     

Molecular Defects in Moroccan Patients with Ataxia-Telangiectasia

Ataxia-telangiectasia (AT) is a rare autosomal recessive disease, affecting neurologic and immune system. Numerous mutations are described in the ATM gene in several populations. However, in Morocco, few data are available concerning this condition. Our main goal is to determine clinical, immunological, and molecular presentation of Moroccan patients with AT. We screened 27 patients, out of 22 unrelated families, for ATM gene mutations. All our patients showed ataxia, ocular telangiectasia, and immunodeficiency, as well as elevated serum alphafetoprotein levels. Mean age at diagnosis was 5.51 years, and consanguinity rate was 81.8 %. Mean age at onset was 2.02 years, and mean time to diagnosis was 3.68 years. We found 14 different mutations in 19 unrelated families, of which 7 were not reported. Our results showed that c.5644C>T mutation was the most common in our series. However, further studies are required to demonstrate a founder effects on ATM gene in Moroccan patients, who showed mutational heterogeneity otherwise. Our data indicate that direct sequencing of coding exons is sufficient for a high detection rate in ATM in Moroccan population.     

Location of the Neuroendocrine Dopamine Neurons in the Monkey Hypothalamus by Retrograde Tracing and Immunostaining*,**

In order to localize neuroendocrine dopamine neurons in the monkey hypothalamus, one female and three male juvenile cynomolgus macaques were each given two or three microinjections (0.2 to 0.3 μl per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion, and vibratomed at 40 pm in the frontal plane. Every 12th section was immunostained with rabbit anti-tyrosine hydroxylase using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine, immunoreactive neurons were easily recognized as brown, immunopositive cell bodies containing more than three distinct dark blue granules, confirmed by electron microscopy to be tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of tyrosine hydroxylase-immunoreactive and neuroendocrine, immunoreactive neurons determined. Although regional and interanimal variations were observed, we estimated that 5,400 of the total 19,000 tyrosine hydroxylaseimmunoreactive neurons in the juvenile macaque hypothalamus were neuroendocrine. When averaged by anatomical region, the suprachiasmatic nuclear groups contained 7% of all immunoreactive neurons (50% were neuroendocrine) and 15% of all neuroendocrine, immunoreactive neurons in these animals. The combined periventricular zones contained 20% of all immunoreactive neurons (more than 50% of ventral and 38% of dorsal were neuroendocrine) and 58% of all neuroendocrine, immunoreactive neurons. The paraventricular nucleus included 50% of all immunoreactive neurons, more than any other nucleus, (3% were neuroendocrine) and 11% of the total neuroendocrine, immunoreactive neurons. The ventral paraventricular nucleus contained only 2% of all immunoreactive neurons (13% were neuroendocrine) and 3% of the total neuroendocrine group. The zona incerta contained 15% of all immunoreactive neurons (0% were retrogradely labeled) but 0% of the neuroendocrine cells. The arcuate nucleus subdivisions contained about 5% of all immunoreactive neurons (more than 60% were neuroendocrine) and 8% of the neuroendocrine population. The ventral hypothalamic tract contained about 1% of all immunoreactive neurons (medially, 63%, and further laterally, 25% were neuroendocrine) and 5% of all neuroendocrine, immunoreactive neurons in these animals. The presence of the retrograde tracer from the median eminence in tyrosine hydroxylase-immunoreactive neurons, combined with knowledge of the location of dopaminergic cell groups, permitted assessment of the A11–A14 dopaminergic neurons which project to the primate infundibulum. Neuroendocrine dopamine neurons occurred predominantly in the All periventricular zones (65% of the total), being greatest around the ventral aspect of the entire third ventricle. They were less numerous in more dorsal regions of All extending up to the level of the paraventricular nucleus. The A12 arcuate (tuberoinfundibular) projection (15% of the total) was not nearly as prominent as All in primates, in contrast to rodents. None of the A13 incertohypothalamic dopamine neurons (0%) projected to the median eminence. The A14 anterior-ventral periventricular region, including the suprachiasmatic nuclear groups, provided the substantial remainder (20%) of all neuroendocrine dopamine neurons. In summary, our results suggest the involvement of a regionally specific dopaminergic system in the hypothalamic control of anterior pituitary hormone secretion in primates. The data also indicate that 75% of all tyrosine hydroxylase-immunopositive neurons do not project to the median eminence, and probably serve other functions. Although the retrograde tracer may not have labeled all neuroendocrine dopamine neurons, it may have identified some dopamine neurons which only interact with other median eminence nerve terminals, or other types of tyrosine hydroxylase-containing, neuropeptidergic neurons which project to the infundibulum. However, considering the known locations of dopaminergic neurons and the large numbers of labeled cells, the results here are a reliable indication of the diverse origins of median eminence-directed dopamine neurons in the juvenile primate.     

DNA微阵列对DMD基因缺失检测的分析

目的:制备简易DNA微阵列检测DMD基因常见外显子缺失,作为一项新技术的方法学摸索,为开发更完善的DMD基因诊断芯片作准备。方法:应用分子克隆的方法扩增DMD基因18个常见易缺失外显子片段,以此作为探针制备出简易DNA微阵列,对DMD患者和健康对照的基因进行检测分析。结果:应用简易DNA微阵列检测出4例DMD患者具有不同程度的外显子缺失,其结果与PCR验证相符。对照满意。结论:DNA微阵列技术适用于DMD基因缺失检测,具有简便、高通量、灵敏等特点。     

The Impairment of Reminiscence Phenomenon in the Percept-Motor Learning in Schizophrenic Patients

Sixteen schizophrenics and 16 months were administered the mirror drawing test twice for 5 minutes, with a rest of 10 minutes intervening. 1. In speed, the score on the last pre-rest trial was not significantly different from that on the first post-rest trial within the schizophrenic group, while within the normal group the difference was significant. 2. In accuracy and task performance, within both the schizophrenic and normal groups, the differences between the two scores were significant. 3. In all three scales, the reminiscence scores, as the difference between the first post-rest and the last pre-rest trial, of the schizophrenic group were significantly lower than those of normal groups. These results showed that the schizophrenic was characterized by low reminiscence scores. It was attempted to explain the schizophrenic in both terms of the inhibition theory and the consolidation theory of reminiscence.     

Location of the Neuroendocrine Gonadotropin-Releasing Hormone Neurons in the Monkey Hypothalamus by Retrograde Tracing and Immunostaining*,**

In order to localize neuroendocrine gonadotropin-releasing hormone (GnRH) neurons in the monkey hypothalamus, four juvenile cynomolgus macaques (one female, three males) were each given two or three microinjections (0.2 to 0.3 μl per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion and vibratomed at 40 μm in the frontal plane. Every 12th section was immunostained with rabbit anti-GnRH using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine GnRH neurons were easily identified in tissue sections as brown, immunostained cell bodies containing more than three distinct, dark blue, tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of GnRH and neuroendocrine GnRH neurons determined. The highest proportion of neuroendocrine GnRH neurons (with projections to the median eminence) occurred in the ventral hypothalamic tract, especially in its medial third (71%), and in the supraoptic decussation just anterior to it. Proportions decreased moving laterally into the middle third (58%) and lateral third (25%) of the ventral hypothalamic tract. Further anterior and lateral, progressively smaller but significant neuroendocrine GnRH contributions were found in the supraoptic nucleus (57%) and lateral hypothalamus (33%), and in the medial preoptic area (26%). Although the medial preoptic area contained a greater percentage of the total GnRH-immunoreactive cell bodies (36%) than the ventral hypothalamic tract (27%), as a whole, the ventral hypothalamic tract contained 60% of the neuroendocrine GnRH neurons compared to only 25% from the medial preoptic area. Large numbers of GnRH cell bodies found in the diagonal band of Broca near the organum vasculosum of the lamina terminalis were not retrogradely labeled. GnRH neurons were not observed in the arcuate nucleus, the few in the paraventricular nucleus were not neuroendocrine, and the contribution from the periventricular zone was negligible. Our results here are the first to identify the neurons giving rise to the neuroendocrine GnRH system in juvenile monkeys. The data indicate that more GnRH neurons close to the infundibulum serve a neuroendocrine (perhaps hypophysiotropic) role than do those in more anterior areas. Furthermore, they suggest that the ventral hypothalamic tract is the most important, and perhaps most influential, neuroendocrine GnRH cell group in primates. The data substantiate the observed autonomy of the medial basal hypothalamus in controlling gonadotropin secretion and menstrual cyclicity in these animals. However, they also infer that perhaps 60% of the GnRH neurons do not project to the primate median eminence, and thus may serve other non-neuroendocrine functions.     

血管回声跟踪技术对高血压伴高脂血症患者颈动脉弹性功能的检测及临床意义

目的 应用血管回声跟踪技术（Echo-Tracking,ET）检测高血压及高脂血症患者的颈动脉弹性功能。方法 选择我院就诊的患者40例,其中高血压患者12例,高脂血症患者12例,高血压伴高脂血症患者16例。同期随机选取正常人10例。应用ET评估颈动脉硬化参数,包括压力应变弹性系数（Ep）、僵硬度（β）、顺应性（AC）、脉搏波传导速度（PWVβ）和增大指数（AI）,通过这些参数了解高血压伴高脂血症患者动脉弹性的变化。结果 高血压伴高脂血症患者Ep、β、PWVβ、AI较正常对照组明显增高,AC明显减小。高血压患者Ep、β、PWVβ、AI较正常人无明显增高,AC无明显减小;高脂血症患者Ep、β、PWVβ较正常人无明显增高,AC无明显减小。结论 应用ET能反映血管的早期病变。高血压伴高脂血症患者颈动脉弹性功能发生了改变,因此,对这些患者加强监测可能对于脑血管疾病的防治有一定的价值。     

克隆外显子探针反向斑点杂交检测DMD基因缺失

目的 :制备检测 DMD基因常见易缺失外显子的核苷酸探针 ,通过反向斑点杂交试验验证其特异性 ,为初步研制 DMD基因诊断芯片作准备。方法 :从健康人外周静脉血白细胞提取基因组 DNA,应用经典 18对引物对 DMD基因常见易缺失外显子进行 PCR扩增。将扩增产物与 p GEM@- T Easy载体连接 ,转化 E.coli JM10 9感受态细胞。克隆目的片段 ,并作测序鉴定。以此为探针进行反向斑点杂交试验。结果 :序列分析表明 ,克隆片段代表 DMD基因 18个常见易缺失外显子 ;以这些片段为探针进行反向斑点杂交 ,其结果与 PCR相符。结论 :克隆的基因片段用作探针在反向斑点杂交试验中显示出较好的特异性 ,可用于 DMD基因常见缺失的检测     

克隆外显子探针反向斑点杂交检测MDM基因缺失

目的：制备检测DMD基因常见易缺失外显子的核苷酸探针，通过反向斑点杂交试验验证其特异性，为初步研制DMD基因诊断芯片作准备。方法：从健康人外周静脉血白细胞提取基因组DNA，应用经典18对引物对DMD基因常见易缺失外显子进行PCR扩增。将扩增产物与pGEM@-T Easy载体连接，转化E.coli JM109感受态细胞。克隆目的片段，并作测序鉴定。以此为探针进行反向斑点杂交试验。结果：序列分析表明，克隆片段代表DMD基因18个常见易缺失外显子；以这些片段为探针进行反向斑点杂交，其结果与PCR相符。结论：克隆的基因片段用作探针在反向斑点杂交试验中显示出较好的特异性，可用于DMD基因常见缺失的检测。     

Immunostaining of calmodulin and aluminium in Alzheimer's disease-affected brains

Previous in vitro studies have shown that Al(3+) binds to calmodulin, inducing alterations in its capability to interact with target proteins, accompanied by loss of immunological recognition by its conformational specific monoclonal antibody CAM1. In spite of the wealth of data of calmodulin action in vitro, little information is available on the possible involvement of this protein in the pathology typical of Alzheimer's disease. In the present study, we investigated calmodulin immunoreactivity in post-mortem human brains affected by Alzheimer's disease, compared with age-matched control brains. Conformational monoclonal antibodies raised against Ca(2+)-calmodulin, namely CAM1 and CAM4, were used in this study for the characterization of calmodulin. Calmodulin immunorecognition by monoclonal antibody CAM1 was found to be lost in cortical tissue sample from brains affected by Alzheimer's disease. This finding leads to the hypothesis of a new, possibly inactive, conformation of the molecule during the disease. On the other hand, CAM4 immunoreactivity was decreased in neurons of brains affected by Alzheimer's disease. Anti-Al(3+) monoclonal antibodies revealed instead more marked aluminium immunoreactivity in the affected brains compared to normal ones. The loss of CAM1 immunoreactivity and the occurrence of large amounts of aluminium suggest an alteration of the active conformation of calmodulin in disease-affected brains. These alterations could be involved in the development of Alzheimer's disease pathology.