抑郁大鼠模型额叶及海马区Rho激酶表达变化的初步研究

目的：观察抑郁大鼠模型急性期额叶及海马区Rho激酶（ROCK）的表达变化,探讨Rho／ROCK通路是否与抑郁症形成有关。方法：建立Wistar大鼠抑郁症模型;在建模成功后24h迅速断头取前脑,在冰浴上分离出双侧前额叶及海马脑组织,应用Westernblot分别测定大鼠额叶及海马脑组织内ROCKⅠ及ROCKⅡ含量。结果：与正常对照组比,抑郁模型大鼠左侧和右侧前额叶ROCKⅠ表达均显著增高（分别P〈0．01,P〈0．05）,以左侧为著;ROCKⅡ表达亦有显著增高（均P〈0．05）。抑郁模型大鼠左右侧海马区域ROCKI表达显著增高（分NP〈0．05,P〈0．01）,以右侧明显;ROCKⅡ表达亦有显著增高（分别P〈0．05,P〈0．01）。结论：ROCKⅠ及ROCKⅡ可能参与了抑郁症形成的病理过程。     

脑源性神经营养因子促进心脏微血管内皮细胞管状形成

背景：心脏微血管内皮细胞在缺血梗死心肌的血管新生中扮演重要角色。最近研究证明：脑源性神经营养因子（BDNF）可促进内皮细胞的存活，诱导血管新生，被认为是一种促血管新生的生长因子，但其促进血管新生的细胞与分子机制尚不甚清楚。 目的： 使用3D体外管状形成模型对脑源性神经营养因子能否促进心脏微血管内皮细胞的管状结构形成进行研究，以揭示BDNF促进血管新生的可能细胞机理。 方法： 经分离的大鼠心脏微血管内皮细胞悬浮于含0.2%甲基纤维素的DMEM培养液（25% FBS）中于非贴壁U型底96孔培养板中培养，使之形成细胞小球，将CMECs小球于含10% FBS的Ⅰ型胶原（1.5 mg/ml）-甲基纤维素（1:1）中混匀后，置37 ℃，5% CO2的培养箱30 min，然后分别加入不同浓度（50 ng/ml、70 ng/ml、100 ng/ml）的脑源性神经营养因子，经培养1天后，对心脏微血管内皮细胞的分枝长度和分枝条数进行分析。 结果： 经分离的大鼠心脏微血管内皮细胞小球在甲基纤维素和Ⅰ型胶原3-D环境中培养24、48小时后，对每个心脏微血管内皮细胞小球管状分枝总长度进行比较发现：24小时后，对照组为2899?5 祄；50 ng/ml BDNF组为3612?54 祄；70 ng/ml BDNF组为4734?82 祄；100 ng/ml BDNF组为5057?72 祄；48小时后：对照组为5349?58 祄；50 ng/ml BDNF组为6085? 祄；70 ng/ml BDNF组为8176?01 祄；100 ng/ml BDNF组为8358?89 祄；不同浓度BDNF处理组的管状分枝总长度分别与对照组比较，均大于对照组，差异有统计学意义（P < 0.05）。对每个心脏微血管内皮细胞小球的分枝总条数进行比较发现：对照组为15条；50 ng/ml BDNF组为19条；70 ng/ml BDNF组为20条；100 ng/ml BDNF组为21条；不同浓度BDNF组的分枝总条数与对照组比较，均大于对照组，差异有统计学意义（P < 0.05）。 结论：脑源性神经营养因子在3D体外管状形成模型中可促进大鼠心脏微血管内皮细胞发芽及管状结构的形成，且脑源性神经营养因子促进心脏微血管内皮细胞管状结构形成的能力，在一定浓度范围内随其浓的升高而呈增强。本研究结果提示：脑源性神经营养因子有效促进心肌血管新生的细胞机理，很有可能是通过有效促进心脏微血管内皮细胞发芽和管状结构的形成。     

脑微血管内皮细胞缺氧模型转化生长因子β1表达与脑溢安的干预

背景：临床及动物实验证实脑溢安能促进脑出血急性期血肿吸收及神经功能恢复，提高生活质量。 目的：观察脑溢安血清对体外缺氧培养的大鼠脑微血管内皮细胞转化生长因子β1 mRNA表达的影响。 方法：用分离培养的大鼠脑微血管内皮细胞移入厌氧培养箱培养18 h建立缺氧损伤模型，并随机分为正常组、模型组、正常血清组及含脑溢安血清组。正常组为同一批正常培养的脑微血管内皮细胞；模型组将正常培养的细胞放入厌氧培养箱内培养18 h；正常血清对照组细胞在培养液中加5%正常血清后，放入厌氧培养箱中培养18 h；脑溢安血清组细胞在培养液中加5%脑溢安血清后，放入厌氧培养箱内培养18 h。 结果与结论：缺氧培养使脑微血管内皮细胞存活数量减少，其表达的转化生长因子β1 mRNA增强，而脑溢安血清能明显增加脑微血管内皮细胞存活数量，降低转化生长因子β1 mRNA表达水平(P < 0.05)。说明脑溢安血清下调转化生长因子β1mRNA的表达可能是其抑制脑微血管内皮细胞的凋亡对缺氧损伤脑微血管内皮细胞的保护作用机制之一。     

肌球蛋白轻链激酶和Rho激酶在兔脑血管平滑肌细胞迁移中的作用

【摘要】目的 探讨肌球蛋白轻链激酶（MLCK）和Rho激酶在颅内血管平滑肌细胞（VSMC）迁移中的作用。方法 体外培养兔大脑中动脉VSMC。构建携带反义MLCK cDNA的腺病毒载体并转染VSMC以降低MLCK表达。应用Rho激酶特异性抑制剂Y-27632来抑制Rho激酶的活性。免疫印迹法测定细胞内MLCK和Rho激酶的表达水平；改良Boyden小室法检测血管紧张素Ⅱ（ATⅡ）诱导的VSMC迁移；甘油凝胶电泳和免疫印迹法检测肌球蛋白轻链（MLC）的磷酸化水平。结果 反义MLCK cDNA转染后，MLCK表达量较LacZ基因转染组下降（84.6±5.8）%（P＜0.01），但ATⅡ刺激引起的MLC磷酸化和VSMC迁移未受明显影响（P均＞0.05）。Y-27632预处理反义MLCK cDNA转染后的VSMC可明显降低基础水平以及ATⅡ刺激引起的MLC磷酸化，并部分阻断VSMC迁移（P均＜0.05）。结论 颅内、外动脉VSMC迁移的分子机制不同，在ATⅡ诱导的颅内VSMC迁移中MLCK作用有限，而Rho激酶可能通过非钙依赖型MLC磷酸化途径发挥重要作用。     

黄芪注射液对体外血管新生的影响*

背景：血管新生受生长因子的影响,黄芪可与血管内皮生长因子具有协同促血管新生的功效,但机制尚不明确。 目的：通过与单一血管内皮生长因子干预比较,观察中药黄芪对体外血管新生的作用机制。 方法：将黄芪注射液及血管内皮生长因子作用于SD大鼠胸主动脉内皮细胞,应用细胞增殖、细胞迁移、小管形成实验观察黄芪注射液对体外血管新生的促进作用,用Western blot实验检测血管内皮生长因子的表达。 结果与结论：黄芪能明显促进大鼠胸主动脉内皮细胞的增殖(P < 0.01),迁移的细胞数增加(P < 0.01),胸主动脉内皮细胞的小管形成数增加(P < 0.01),并明显促进内皮细胞血管内皮生长因子的表达(P < 0.01)。说明黄芪能明显促进内皮细胞增殖、细胞迁移、小管形成,具有显著的促进体外血管新生作用,其机制可能是通过增加血管内皮生长因子的表达而实现的。     

HGF对人胶质瘤新生血管形成的影响研究

目的 探讨肝细胞生长因子(HGF)在人胶质瘤新生血管形成中的作用及可能机制.方法 中山大学附属第一医院病理科白2002年1月至2008年6月共确诊胶质瘤患者72例,其中Ⅰ级19例,Ⅱ级18例,Ⅲ级20例,Ⅳ级15例,免疫组化染色检测胶质瘤组织HGF、CD34的表达.体外常规培养胶质瘤细胞株U87MG,将荧光标记的HGF si-RNA(si-HGF)特异性干扰序列转染至细胞内为转染si-HGF组,同时设正常对照组和阴性转染组作为对照；Western blotting检测转染后细胞HGF、血管内皮生长因子(VEGF)的表达；将3组细胞悬液接种于鸡胚尿囊膜(CAM),5d后观察各组移植瘤周围血管形成情况. 结果 低级别胶质瘤(Ⅰ级、Ⅱ级)HGF阳性率低于高级别胶质瘤(Ⅲ级、Ⅳ级),HGF阳性胶质瘤中微血管密度(MVD)高于HGF阴性胶质瘤,差异均有统计学意义(P＜0.05).Western boltting检测结果显示,与正常对照组和阴性转染组比较,转染si-HGF组细胞HGF的表达下降约76％,VEGF的表达下降约53％；移植瘤实验显示转染si-HGF组新生血管数、血管面积及血管生成面积比均低于正常对照组和阴性转染组组,差异有统计学意义(P＜0.05). 结论 HGF与胶质瘤新生血管的形成关系密切,提示其有可能成为今后抗肿瘤血管生成的一个靶点.     

L6大鼠成肌细胞胰岛素信号传导通路中磷脂酰肌醇3激酶、蛋白激酶B和葡萄糖转运蛋白4的表达**★

背景：血管紧张素Ⅱ可损伤胰岛素信号中的下游信号分子引起胰岛素抵抗，但其机制不清。 目的：观察血管紧张素Ⅱ对L6大鼠成肌细胞胰岛素信号传导通路中磷脂酰肌醇3激酶、蛋白激酶B和葡萄糖转运蛋白4的影响。 方法：L6细胞培养及诱导分化肌管，根据干预措施不同实验分为对照组、胰岛素组、胰岛素+血管紧张素Ⅱ组及胰岛素+血管紧张素Ⅱ+H89组。采用RT-PCR检测4组磷脂酰肌醇3激酶、蛋白激酶B mRNA表达，免疫荧光检测胰岛素受体底物1、酪氨酸磷酸化胰岛素受体底物1、葡萄糖转运蛋白4表达。 结果与结论：胰岛素组、胰岛素+血管紧张素Ⅱ组及胰岛素+血管紧张素Ⅱ+H89组的磷脂酰肌醇3激酶mRNA表达均较对照组显著升高(P < 0.05)。各组间蛋白激酶B mRNA表达差异无显著性意义(P > 0.05)。相比对照组，其余3组间胰岛素受体底物1、酪氨酸磷酸化胰岛素受体底物1和葡萄糖转运蛋白4(膜蛋白)表达均升高(P < 0.05)；胰岛素+血管紧张素Ⅱ+H89组酪氨酸磷酸化胰岛素受体底物1和葡萄糖转运蛋白4表达低于胰岛素组但高于胰岛素+血管紧张素Ⅱ组(P < 0.05)。结果显示，血管紧张素Ⅱ在骨骼肌细胞中通过JAK2-PKA通路引起胰岛素下游信号传导受阻，葡萄糖转运蛋白4表达减少，葡萄糖转运障碍，进而导致胰岛素抵抗。     

细胞外信号调节激酶1/2与Rho激酶作用对脑梗死后神经血管单元的影响

目的研究脑梗死后细胞外信号调节激酶1/2(ERK_(1/2))和Rho激酶(ROCK)两条信号通路通过相互作用激活下游效应分子多聚ADP核糖聚合酶-1(PARP-1)来调控脑梗死后神经血管单元。方法该实验分为两个部分:35只SD大鼠随机分为假手术组(s)和脑梗死组(M),脑梗死组根据脑梗死后时间不同又分为1h、3h、12h、24h、3d和7d六个亚组,分别采用westem blot检测假手术组及脑梗死组各亚组ERK_(1/2)、ROCK蛋白表达水平。35只SD大鼠随机分为对照组、模型组、U0126组、Fasudil组和U0126+Fasudil组,分别检测神经功能、脑梗死面积以及ROCK、ERK_(1/2)及PARP-1的蛋白表达水平。结果假手术组各个时间点总ERK_(1/2)/2和p-ERK_(1/2)表达相同。脑梗死组总ERK_(1/2)表达不变,p-ERK_(1/2)表达先降低后升高,24h时达最高峰。脑梗死组ROCK的表达随时问的延长逐渐升高,12h表达达高峰,随后表达下降。模型组与对照组相比,p-ERK_(1/2)、ROCK及PARP-1的表达显著提高(P0.05);与模型组相比,Fasudil组p-ERK_(1/2)的表达下降(P0.05),而U0126组ROCK表达无变化(p0.05),Fasudil组、U0126组及Fasudil组+U0126组PARFP-1的表达显著下降(P0.05),其中以U0126+Fasudil组下降最为显著。结论 ERK_(1/2)和ROCK都参与了脑梗死后脑组织的损伤,ERK_(1/2)可能作为ROCK的下游效应分子,与ROCK共同调节PARP-1的表达进而调控脑梗死后神经血管单元的存亡。     

微血管内皮细胞诱导骨髓间充质干细胞向心肌样分化及其移植的可行性

背景：骨髓间充质干细胞通过再生心肌细胞在心肌损伤性疾病治疗方面突显较大潜力,但目前仍未找到一种理想的诱导方式。 目的：探讨微血管内皮细胞诱导大鼠骨髓间充质干细胞向心肌细胞分化的潜能,并分析其移植的可行性。 设计、时间及地点：细胞学体外观察及体内移植实验,于2008-07/2009-02在重庆市神经病学重点实验室完成。 材料：清洁级二三月龄健康雄性SD大鼠16只,购自重庆医科大学实验动物中心 方法：孔径0.4 µm的transwell小室,置入培养板孔构成双室培养体系。取SD大鼠1只,Ficoll密度梯度+贴壁筛选法分离培养骨髓间充质干细胞,组织块法分离培养微血管内皮细胞。培养瓶中融合至50%生长良好的微血管内皮细胞,每3 d更换培养液,收集更换液过滤即制成条件培养液。体外实验分为4组：直接接触组、双室培养组、条件培养液组、培养液对照组,观察各种培养条件对骨髓间充质干细胞的心肌诱导效应。取SD大鼠15只建立心肌梗死模型,冠状动脉结扎后第6天任取1只大鼠明确心肌梗死建模是否成功,其余大鼠随机分为细胞移植组8只、模型对照组6只。造模1周后,细胞移植组将在双室模型中与微血管内皮细胞共培养5 d的骨髓间充质干细胞调整浓度为109 L-1,通过鼠尾静脉缓慢注入1 mL,模型对照组予等量DMEM,观察4周。 主要观察指标：体外实验通过免疫荧光染色检测心肌标志物TnI、α-actin的表达;采用心电图、荧光镜检、苏木精-伊红染色等方式评价体内移植的安全性及可行性。 结果：直接接触组、双室培养组、条件培养液组部分骨髓间充质干细胞心肌标志物TnI、α-actin阳性表达,荧光镜下胞浆发绿光(或红光),前2组诱导率基本相似(P > 0.05),且均明显高于条件培养液组(P < 0.05);培养液对照组细胞未向心肌样细胞分化。细胞移植后两组大鼠均存活良好,未出现死亡及恶性心律失常,4周后细胞移植组心脏冰冻切片荧光镜检显示心肌内有少量胞核蓝染的细胞,提示干细胞成功归巢,结合苏木精-伊红染色,归巢细胞位于心肌梗死灶周边区域,呈单个或小团聚集;而模型对照组心肌切片未见胞核蓝染的细胞。 结论：微血管内皮细胞能够通过旁分泌途径诱导骨髓间充质干细胞向心肌样细胞分化,经微血管内皮细胞预处理过的骨髓间充质干细胞移植入大鼠体内是安全的,可成功归巢到大鼠心肌梗死灶及周边区。     

BMSCs-完全脱钙骨基质体外诱导构建组织工程纤维软骨

背景：半月板损伤后自行修复能力有限，组织工程技术构建纤维软骨组织为半月板损伤后修复开辟了一条新的途径。 目的：观察猪骨髓间充质干细胞与完全脱钙骨基质在体外诱导培养成纤维软骨组织并检测其凋亡。 方法：采用全骨髓培养法分离培养猪骨髓间充质干细胞，取第1代骨髓间充质干细胞并将细胞密度调整到1?07 /ml，均匀接种到完全脱钙骨基质上，37℃孵育4 h后以含有TGF-β1、IGF-Ⅰ、地塞米松、抗坏血酸及10%FBS的高糖DMEM诱导培养液培养作为试验组，单层诱导培养BMSCs作为对照组Ⅰ，单层基础培养的BMSCs作为对照组Ⅱ，空白完全脱钙骨基质为对照组Ⅲ，在培养第1、2、3、4周分别进行大体形态观察以及Ⅰ、Ⅱ型胶原及聚集蛋白聚糖(Aggrecan)的RT-PCR定性检测；培养4周样本分别进行凋亡检测。 结果与结论：试验组可检测到Ⅰ型胶原的持续表达，Ⅱ型胶原及Aggrecan的表达逐渐增加。对照组Ⅰ可检测到Ⅱ型胶原及Aggrecan的表达及Ⅰ型胶原的持续表达，但表达量远低于试验组；培养4周样本凋亡检测显示试验组凋亡高于对照组Ⅰ，对照组Ⅰ与对照组Ⅱ凋亡无差异。TGF-β1、IGF-Ⅰ体外诱导培养的MSCs-完全脱钙骨基质可以表达特异性软骨基质，三维环境培养细胞外基质的表达量明显高于单层诱导培养；静态培养系统对三维培养的细胞凋亡有影响。     

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.