颈淋巴阻滞对大鼠血脑屏障通透性的影响

目的: 探讨大鼠颈淋巴阻滞后血脑屏障(BBB)通透性的变化。方法: 手术组大鼠采用Casley-Smith法阻滞颈淋巴,对照组大鼠不摘除颈淋巴结和不结扎淋巴管,只将它们分离暴露。每组于术后1、3、5、7、14、21 d,通过观察血清中S-100B的浓度、电镜观察镧离子示踪剂和BBB超微结构变化来确定BBB通透性的改变。结果: 各组血清中S100B浓度变化无显著差异,提示BBB没有破坏;电镜观察:对照组与手术组的超微结构变化相同,各组术后各时点血管腔外没有发现镧离子。结论: 颈淋巴阻滞对BBB通透性没有显著影响。     

Process of differentiation of cerebellar Purkinje neurons in the chick embryo

Summary The microscopic and ultrastructural differentiation of Purkinje neurons has been studied in 40 chicken embryo cerebella, from the 10th incubation day to hatching, and the transverse diameter of the cell body measured, for each developmental stage, on 30 electron micrographs of sagittally cut Purkinje cells. The developing Purkinje cell bodies, bipolar, at first, given the presence of two processes emerging from the opposite poles of the oval perikaryon, grow progressively in size. After the 12th incubation day, they develop a branched dendritic tree, and, shortly before hatching time, the cells acquire the characteristic flask or pear-shaped configuration. On the 10th incubation day, microtubules are already detectable together with Golgi complexes and a few vesicles of rough endoplasmic reticulum; on the 14th incubation day, RER cisterns are recognizable in the supranuclear cytoplasm, later extending into the whole perikaryon, and attaining their definitive distribution by the 18th incubation day. Pinocytotic and coated vesicles, as well as subsurface cisterns are seen during the whole embryonic life. In the earliest stages of development, three distinct types of junctional contacts between Purkinje cells and surrounding axons are described, and their functional role in relation to synaptogenetic processes is discussed. Beginning with the 16th incubation day, some Purkinje neurons undergo degenerative changes similar to those described in other types of neurons of the central and peripheral nervous system.     

Onset of elastogenesis and downregulation of smooth muscle actin as distinguishing phenomena in artery differentiation in the chick embryo

During development, the arterial system is grossly divided into elastic and muscular vessel types. Apart from local environmental factors, it has been suggested that vascular smooth muscle cell origin (mesoderm or neural crest) is involved in this, as yet poorly understood, arterial differentiation. We describe differentiation of the thoracic arterial system in the chick embryo, using immunohistochemical techniques staining for muscle-specific actin, vinculin and desmin and histological staining to visualise elastin. The initial developmental stages of the vessel wall in all arteries appeared to be highly similar, with all arteries showing peri-endothelial actin and vinculin staining. Major alterations did not occur until the start of elastogenesis, which coincided with complete loss of actin staining from the proximal part of the great arteries. Later in development, however, actin was re-expressed in a subpopulation of medial cells, which also expressed vinculin and desmin. Concomitantly another, nonmuscular, cell type became evident in the great arteries. Transient loss of actin expression and segregation of very distinct cell populations occurred only in vessels prone to elastic development and known to receive a neural crest contribution. In contrast, arteries that developed a muscular phenotype never lost the initially acquired peri-endothelial actin expression. We also show a significant difference in the organisation of elastic fibres between elastic vessels that contain neural crest derivatives and those that do not. The ductus arteriosus still presents as an enigma in the sense that it is the only part of the pharyngeal arch complex that develops a muscular phenotype.     

Application of cultured endothelial cells of the brain microvasculature in the study of the blood-brain barrier

Summary The endothelial cells of the brain microvasculature provide both physical and enzymatic barriers to the exchange of molecules between the extracellular fluid environment of the brain and the systemic circulation. To better understand these barrier properties and the factors that influence them at the cellular level, an in vitro model of the blood-brain barrier has been developed using primary cultures of bovine brain microvessel endothelial cells. The focus of the present paper is on the procedures associated with the isolation, culturing, and maintenance of bovine brain microvessel endothelial cells. Experimental applications of the cell culture model in studying the blood-brain barrier and efforts directed at improving the current cell culture model are also discussed.     

The interferon-induced expression of APOBEC3G in human blood-brain barrier exerts a potent intrinsic immunity to block HIV-1 entry to central nervous system

In the human genome, the APOBEC3 gene has expanded into a tandem array of genes termed APOBEC3A-H. Several members of this family have potent anti-HIV-1 activity. Here we demonstrate that APOBEC-3B/3C/3F and -3G are expressed in all major cellular components of the CNS. Moreover, we show that both interferon-alpha (IFN-alpha) and IFN-gamma significantly enhance the expression of APOBEC-3G/3F and drastically inhibit HIV-1 replication in primary human brain microvascular endothelial cells (BMVECs), the major component of blood-brain barrier (BBB). As the viral inhibition can be neutralized by APOBEC3G-specific siRNA, APOBEC3G plays a key role to mediate the anti-HIV-1 activity of IFN-alpha and/or IFN-gamma. Our findings suggest that, in addition to the restriction at viral entry level, the restriction from APOBEC3 family could account for the low-level replication of HIV-1 in BMVECs. The manipulation of IFN-APOBEC3 signaling pathway could be a potent therapeutic strategy to prevent HIV invasion to central nervous system (CNS).     

Perivascular astrocytes and endothelium in the development of the blood-brain barrier in the optic tectum of the chick embryo

The role played by perivascular astrocytes in neural vessel maturation was investigated in microvessels of the chick embryo optic tectum. Three-dimensional reconstructions and quantitative analyses were made, and permeability was studied. On embryonic days 14–16, 12.5% of the microvessel wall is surrounded by astrocyte endfeet which, in most cases (82%), are located under endothelium junctions; the latter, at this stage, partly prevent the extravascular escape of the marker horseradish peroxidase. On days 18–21, the astrocyte processes form a nearly complete perivascular sheath enveloping 96% of the microvessel perimeter; the junctions of the endothelial cells are much wider and impermeable owing to extensive fusion of the endothelial plasma membranes. This investigation suggests a close relationship between the perivascular arrangement of glia and differentiation of the endothelium tight junctions and indicates that the morphofunctional maturation of the latter takes place progressively during the prenatal organogenesis of the chick central nervous system.     

纤维蛋白对大鼠脑血管内皮细胞白细胞介素6表达的影响

目的：观察纤维蛋白对离体培养的大鼠脑血管内皮细胞白细胞介素6（IL-6）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检测脑血管内皮细胞中IL-6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IL-6水平。结果：加入不同浓度（0.03 g/L、0.1 g/L、0.3 g/L和1.0 g/L）的纤维蛋白24 h后，1.0 g/L纤维蛋白组的培养基中IL-6水平显著增高（P<0.01）；然后加入1.0 g/L纤维蛋白2、4、8、24 h，培养基中的IL-6水平均显著升高（P<0.05）；而加入不同浓度的胶原蛋白不引起IL-6水平的明显变化；real-time PCR结果显示，脑血管内皮细胞IL-6 mRNA的上调呈现出剂量和时间依赖性增加。结论：纤维蛋白是血管内皮细胞活化剂，可以上调大鼠脑血管内皮细胞中IL-6的表达，在蛋白和mRNA均呈现出剂量和时间依赖性增加。     

高糖对脑微血管内皮细胞抗氧化酶系统的影响

探讨高糖刺激时脑微血管内皮细胞锰超氧化物歧化酶及其他抗氧化酶的基因表达改变。通过体外培养小鼠脑微血管内皮细胞系bEnd.3;H2DCF荧光法检测高糖刺激下脑微血管内皮细胞短期内氧自由基（ROS）生成,RT-PCR检测高糖刺激下内皮细胞铜锌超氧化物歧化酶（SOD1）、锰超氧化物岐化酶（MnSOD）、脂质过氧化物酶（GPX）及触酶（CAT）的表达,并通过werstern blot法检测内皮细胞内MnSOD蛋白水平。结果表明：高糖刺激下脑微血管内皮细胞短期内氧自由基（ROS）产生明显增多并持续增高;高糖刺激10小时抗氧化酶MnSOD,GPX,CAT的mRNA水平无明显变化,SOD1 mRNA水平略有增高;SOD1、MnSOD的mRNA水平在高糖刺激24小时后有明显增加,但是MnSOD蛋白水平在高糖刺激48小时内并无明显增加;抗氧化剂白藜芦醇或表没食子儿茶素没食子酸酯（EGCG）不能上调MnSOD蛋白水平。以上数据表明,高糖刺激能诱导细胞内多种抗氧化酶包括MnSOD基因表达上调,但也能抑制MnSOD蛋白水平的增加。     

瓦勒变性坐骨神经段对大鼠BMSCs向SCs分化的影响

 目的:通过观察瓦勒变性坐骨神经段对大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）增殖、分泌功能以及向施万细胞（Schwann cells, SCs）分化的影响,探讨其在BMSCs向SCs分化中的作用及可能的机制。方法:采用全骨髓贴壁法分离、培养SD大鼠BMSCs,并采用免疫荧光法鉴定。应用Transwell建立坐骨神经段与BMSCs双层培养体系,将实验分为瓦勒变性坐骨神经段与BMSCs联合培养组（A组）、正常坐骨神经段与BMSCs联合培养组（B组）和BMSCs单独培养组（C组）。倒置相差显微镜观察联合培养过程中BMSCs 形态变化;免疫荧光染色检测联合培养第7天各组BMSCs表达S-100情况;联合培养第0、1、4、7、11、14天,利用细胞计数法绘制各组BMSCs生长曲线,ELISA法检测各组培养液上清中神经生长因子（nerve growth factor, NGF）含量,实时荧光定量PCR检测各组BMSCs中S-100 mRNA表达情况。结果:成功分离培养BMSCs,免疫荧光鉴定BMSCs呈CD29、CD44和CD90表达阳性。联合培养第7天倒置相差显微镜观察可见,A组BMSCs胞体回缩,呈梭形,并带有突起,形态类似SCs;B、C 组大部分BMSCs 形态无明显变化。联合培养第7天免疫荧光染色示,A、B、C 组 BMSCs S-100阳性表达率分别为31.1%±2.9%、16.2%±1.7%和0.42%±0.07%,A组阳性表达率明显增高（P<0.05）。各组BMSCs生长曲线均近似“S”形,从第4天开始,A组BMSCs增殖速度明显快于B、C组（P<0.05）;ELISA 法检测示,A 组NGF含量呈时间依赖性增加,于联合培养第7天达高峰,随后呈下降趋势。B、C 组NGF含量随共培养时间延长有所增加,但显著低于A组（P<0.05）;实时荧光定量PCR检测示,联合培养第4、7、11、14天,A组S-100 mRNA表达明显高于B、C组（P<0.05）。结论: 瓦勒变性坐骨神经段能有效促进大鼠BMSCs增殖并诱导BMSCs向SCs样细胞分化,联合培养过程中NGF可能参与BMSCs向SCs分化的调控。     

Uptake across the blood-brain barrier and tissue distribution of enterostatin after peripheral administration in rats

Enterostatin, the N-terminal activation pentapeptide of procolipase that is produced by the pancreas, reduces food intake from high-fat diet when injected either peripherally or centrally to rats. We investigated uptake across the blood-brain barrier (BBB) and tissue distribution of enterostatin by giving radioactive-labeled enterostatin (3H-VPDPR) intravenously. Low levels of 3H-VPDPR were detected in many areas of the brain, with greatest radioactivity in the frontal cortex, hippocampus and cerebellum. Radioactivity was found in the plasma and all tissues, with the highest amount detected in the pancreas.     

Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation

The role of astrocytes in microglia-induced neuronal death remains controversial. In this study, astrocytes and astrocyte-derived conditioned media (ACM) supported the survival of dopaminergic neurons, and the former was more effective than the latter. In the presence of astrocytes, low concentrations of LPS enhanced the survival of dopaminergic neurons, while high concentrations attenuated survival. LPS dramatically induced astrocytes to secrete IL-6 in a dose-dependent manner with no effect on secretion of GDNF. Neuron–astrocyte cultures had highest secretion of GDNF, followed by ACM-treated neuron-enriched cultures. After neuron–astrocyte cultures treated with IL-6-neutralizing antibody, both effects of the enhanced and attenuated survival of dopaminergic neurons were abolished. Our results indicate that astrocytes play a protective role in the LPS-induced damage of dopaminergic neurons in certain circumstances, and the interaction between astrocytes and dopaminergic neurons may enhance the protective effect of astrocytes. Suitable activation of astrocytes increases the protective effect while excessive activation attenuates it, and IL-6 might medicate this dual action. The underlying mechanisms related to the secretion of GDNF and proinflammatory factors warrant further investigation.     

胚胎干细胞向成骨、软骨细胞定向分化研究进展

骨、软骨组织工程的热点之一是寻找合适的种子细胞.胚胎干细胞因具有全能性和无限增殖的能力有望成为组织工程中的种子细胞新来源.主要介绍了胚胎干细胞定向诱导分化为成骨、软骨细胞研究所取得的进展,并展望了胚胎干细胞作为组织工程种子细胞的前景和所面临的困难.     

骨髓间充质干细胞对大鼠胶质瘤细胞增殖的影响

目的研究骨髓间充质干细胞（BMSC）对大鼠脑胶质瘤C6细胞增殖的影响,并且探讨其相关机制。方法提取SD大鼠BMSC,进行体外培养、扩增。应用MTt比色法检测不同浓度BMSC上清液对C6细胞系增殖的抑制作用。用Transwell小室将BMSC与C6细胞进行双层培养,以HE染色法检测C6细胞形态变化。用划痕实验检测细胞迁移情况。结果MTF结果显示BMSC上清液对C6细胞有抑制作用,120h后1：8、1：4、1：2的BMSC上清稀释液、BMSC上清原液培养组的生长抑率分别为17．1％、26．0％、39．9％、43．1％;与BMSC进行双层培养后的C6细胞其形态发生了明显改变,由圆形或多角形变为长梭形,细胞包体伸出长突起;划痕实验显示在36h时对照组迁移率为100％,划痕完全愈合,而BMSC上清组划痕未见全愈合,迁移率为82％。结论①BMSC上清液能抑制胶质瘤C6细胞的恶性增殖,而且抑制效应呈剂量依赖性;②BMSC对C6的运动和迁移产生了抑制作用,从而降低了其恶性侵袭程度。     

脑源性神经营养因子基因修饰的胚胎大鼠皮质神经干细胞及其在体外的分化

目的 探讨脑源性神经营养因子（BDNF）基因修饰大鼠脑皮质神经干细胞（NSCs）后BDNF的表达,并观察其对NSCs体外诱导分化的影响。 方法 体外分离、培养大鼠胚胎脑皮质神经干细胞并鉴定;构建BDNF基因重组质粒,非脂质体法转染NSCs,实验分为pcDNA3-1- BDNF组、pcDNA3-1组和未转染NSCs组。免疫细胞化学技术和RT-PCR检测转染后BDNF蛋白和mRNA的表达;在含5％胎牛血清的分化培养基中诱导分化,βIII微管蛋白、神经胶质纤维酸性蛋白（GFAP） 和突触素( SYP)免疫细胞化学染色对分化细胞进行鉴定,并观察pcDNA3-1-BDNF转染NSCs分化过程中SYP的表达变化。 结果 体外培养获得巢蛋白（nestin）阳性的NSCs。成功构建BDNF基因重组质粒,免疫细胞化学和RT-PCR检测均显示,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs的BDNF表达明显增强（P<0.05）。pcDNA3-1组和NSCs组比较,无显著性差异（P＞0.05）。分化后第4天,各组细胞均可分化为Βiii-微管蛋白阳性和GFAP阳性细胞,pcDNA3-1-BDNF组可见少量SYP阳性表达细胞。分化后第7天,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs分化为βIII微管蛋白阳性神经元的比例明显增高,有显著性差异（P<0.05）,SYP阳性细胞数增多,表达增强。 结论 BDNF转染NSCs具有体外分泌BDNF的能力,能够促进NSCs定向分化为神经元,SYP表达增强,可能在促进神经元之间的突触发生中发挥作用。     

A Golgi study on the early sequence of differentiation of ganglion cells in the chick embryo retina

Summary An examination of retinal structure in chick embryos, impregnated with the Golgi-Stensaas method between 2 and 6 days of incubation, discloses, on the one hand, a uniform typology of the proliferating ventricular cells, the pre- and postmitotic forms of which were tentatively identified; on the other hand, postmitotic neuroblasts are evidenced in the stages of differentiation previous to the growth of their neurites.In the earliest embryos (up to 51/2 days of incubation), all cells that detach from the ventricular lining to differentiate as neurons do so while the ventricular cell precursor has an interphasic configuration. This means that, although they free themselves from the scleral attachment site, they keep for a while a vitreal attachment. The vitreally-attached endfeet subsequently transform into axonal growth cones, sprouting filopodia and lamellipodia. While the axons grow towards the optic nerve head, cell bodies and remaining scleral processes are progressively retracted inwards, leading to the appearance of typical ganglion cells.After 51/2 days of incubation, a great number of postmitotic neuroblasts detach while still in the G₁ phase of the ventricular cell cycle. Those of them that show the longest leading processes become also ganglion cells, after their leading tip has acquired a growth cone configuaration and has bent into the optic fiber layer.These results on early mechanisms of ganglion cell genesis are discussed in relation to data in the literature, and a simple hypothesis is offered which explains the biphasic pattern in which presumptive ganglion cells detach from the ventricular lining of the chick retina.     

雷米普利对充血性心力衰竭患者血循环内皮细胞及脑钠素的影响

目的探讨充血性心力衰竭(CHF)时血循环内皮细胞计数(CEC)、血浆脑钠素(BNP)、血浆内皮素-1(ET-1)及6-酮-前列腺素F1α(6-Keto-PGF1α)水平的变化及雷米普利的干预效应.方法25例CHF患者服用雷米普利两周,并检测用药前后自身外周血循环CEC数量、BNP、ET-1及6-Keto-PGF1α的含量,20例健康体检者为对照组.结果CHF患者CEC数量、血浆ET-1及BNP水平明显增高,6-K-PGF1α明显降低(P均＜0.01).雷米普利治疗可使CEC数量、血浆ET-1及BNP水平明显降低及6-Keto-PGF1α含量显著增加(P＜0.01).结论心力衰竭时存在血管内皮细胞受损,而雷米普利具有保护血管内皮细胞及改善心脏功能的作用.     

A developmental study on the expression of PDGFαR immunoreactive cells in the brain of postnatal rats

The platelet-derived growth factor-α receptor (PDGFαR) has been found specifically expressed in oligodendrocyte precursor cells (OPCs), whereas another membrane protein, NG2, has been widely applied to characterize developing and matured OPCs. In order to investigate whether PDGFαR expression is consistent to NG2 in identifying OPCs, we utilized techniques of immunohistochemistry and Western blot to study the PDGFαR expression and distribution in rat postnatal brain from a series of ages P0 (postnatal day 0) to P540, and further compared it with NG2. Results showed that PDGFαR immunoreactive (PDGFαR+) cells existed in both the gray and white matter of the postnatal rat brain, although these cells displayed different features in distinct regions and developmental stages. PDGFαR did not express in oligodendrocytes, astrocytes or neurons (indicated by non-co-localization with CC1 and NF200, respectively). Western blot analysis revealed that the expression of PDGFαR in the cerebral cortex and hippocampus increased from P0 to P7 and then decreased gradually. PDGFαR+ cells displayed similar characteristics as of NG2+ cells in the morphology, distribution and electrophysiology. Like NG2+ cells, the density of PDGFαR+ cells had an increase at P7 and a late age-dependent decline, except a lower value from P7 to P540 in cerebral cortex, hippocampus and corpus callosum. PDGFαR+ cells exhibited number consistent with NG2+ cells at early developmental stages and were approximately 75% as numerous as NG2+ cells at old age. PDGFαR+/NG2− cells were not found. In conclusion, our findings suggest that both PDGFαR and NG2 are markers of developing OPCs. PDGFαR is specific to the NG2+ OPCs and mainly plays an important role at early developmental stages of OPCs. Aging had an effect on the morphological feature, number and developmental regulation of OPCs in rat CNS. However, further work will be necessary to determine if PDGFαR−/NG2+ cells may still maintain the biological characteristics of OPCs or they are other subpopulation of OPCs.     

脑源性神经营养因子体外诱导神经干细胞向神经元分化

目的观察脑源性神经营养因子(BDNF)对新生SD大鼠海马神经干细胞在体外分化为神经元的作用。方法取新生SD大鼠海马组织,以无血清培养技术培养获得神经干细胞,在BDNF诱导下让其在体外分化,7d后,通过免疫荧光技术结合图像分析技术来观察分化所得神经丝(neurofilament,NF)抗原阳性神经元的比率及其最长突起的长度。结果BDNF组分化所得细胞NF阳性率为13.66%,神经元突起长度为(146.27±26.30)μm,对照组分化所得细胞NF阳性率为9.38%,神经元突起长度为(117.00±23.98)μm,两组结果有显著性差异。结论BDNF能提高新生SD大鼠海马神经干细胞体外定向分化为神经元的比率,并且能刺激新生神经元突起的生长。     

Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo

Summary The role of a notochord fragment on the origin of an additional floor plate area in the neural tube is investigated by quantitative morphological methods. In 1.5 to 2 day chick embryos a notochordal fragment was implanted in close apposition to the lateral wall of the neural groove in the region between prospective wing and leg bud. At 4 days, adjacent to the implant a distinct area of the neural wall was present, which resembled the natural floor plate with respect to its thickness, the abluminal location of elongated nuclei and the absence of neuroblasts. The mitotic density of this area was reduced. This additional floor plate was distinct when the experiment was performed at 1.5 days but was hardly recognizable when it was carried out at 2 days.From these results it is concluded that a) the notochord induces floor plate like structures and diminishes proliferation, and b) that the period of floor plate induction by the notochord is very restricted.