Mash1在室管膜前下区神经干细胞向神经元分化中的作用

目的研究Mashl在室管膜前下区(SVZa)神经干细胞向神经元分化中的作用。方法体外分离培养新生0 d昆明小鼠的SVZa神经干细胞,原位杂交检测Mashl在SVZa神经干细胞的表达;构建Mashl与绿色荧光蛋白(GFP)正义、反义融合蛋白表达质粒,转染SVZa神经干细胞,GFP活体荧光标记SVZa神经干细胞;采用细胞计数和流式细胞仪检测在Mashl作用下SVZa神经干细胞分化为神经元的比例。结果体外培养的新生0 d昆明小鼠的SVZa神经干细胞Mashl原位杂交检测阳性;Mashl-GFP 组SVZa神经干细胞分化为神经元的比例明显高于空白对照组;Mashl-GFP-组 SVZa神经干细胞分化为神经元的比例明显低于空白对照组。结论体外培养的新生0 d昆明小鼠的SVZa神经干细胞表达Mashl;Mashl可以促进SVZa神经干细胞向神经元方向分化。     

人脂肪组织来源的神经干细胞移植对大鼠局灶性脑缺血再灌注后细胞凋亡及Bcl-2、Bax蛋白表达的影响

目的 探讨人脂肪组织来源的神经干细胞移植对大鼠局灶性脑缺血再灌注后细胞凋亡及Bcl-2、Bax蛋白表达的影响.方法 线栓法制作大鼠大脑中动脉缺血2 h再灌注模型.60只健康雄性SD大鼠随机分为4组:正常对照组(6只),假手术组(6只),缺血对照组(24只)和移植治疗组(24只);后2组又分为再灌注7 d、14 d、21 d、28 d组(各6只).体外培养脂肪基质细胞,诱导分化为神经干细胞.造模成功后24h,移植治疗组经尾静脉移植人脂肪组织来源的神经干细胞悬液(细胞浓度为2×106/ml),缺血对照组经尾静脉注射生理盐水,假手术组不做任何处理.TUNEL法检测细胞凋亡,免疫组化SABC法检测Bcl-2、Bax表达.结果 与缺血对照组比较,移植治疗组各时间点的细胞凋亡数均明显减少(均P＜0.01),Bcl-2阳性细胞数明显增高(均P＜0.01),Bax阳性细胞数明显减少(P＜0.05～0.01).结论 人脂肪组织来源的神经干细胞可能通过上调Bcl-2蛋白表达、下调Bax蛋白表达,减少局灶性脑缺血细胞凋亡;对脑缺血再灌注损伤后的神经细胞起保护作用.     

缺血性卒中大鼠海马的Notch信号通路功能的动态变化

目的 研究Notch通路在局灶性缺血性脑卒中大鼠海马的动态变化,探讨该通路对缺血性卒中后内源性神经前体细胞再生的调控作用.方法 以大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)法建立大鼠局灶性脑缺血模型,通过Western blotting法以及Real-time RT-PCR法,于术后19 d、28 d检测海马Notch通路下游靶基因Hes 1、Hes 5的蛋白和mRNA表达.结果 术后19 d,MCAO组Hes 1、Hes 5蛋白及mRNA表达均分别高于对照组,差异有统计学意义(P＜0.01,P＜0.05),28 d时MCAO组Hes 1、Hes 5的蛋白及mRNA表达下降,较之对照组差异有统计学意义(均P＜0.05),而且低于19 d时Hes 1、Hes 5的表达,差异有统计学意义(均P＜0.01).结论 卒中后的内源性神经再生机制中可能有Notch通路活性动态变化的机制参与,卒中后期Notch通路活性的显著下调可能是促进增殖的神经前体细胞向神经元方向分化的重要分子机制.     

新生大鼠海马神经干细胞表达趋化因子受体CX3CR1的体外研究

目的 探讨体外培养神经干细胞是否表达趋化因子受体CX3CR1.方法 采用无血清方法分离、培养新生大鼠海马神经干细胞,细胞免疫荧光方法检测神经干细胞标志巢蛋白(nestin)表达及干细胞多向分化为神经元及胶质细胞的能力,后通过细胞免疫荧光及RT-PCR方法检测神经干细胞表达趋化因子受体CX3CR1的情况.结果 体外培养新生大鼠海马神经干细胞呈nestin阳性.可分化为神经丝蛋白200(NF200)阳性的神经元、胶质纤维酸性蛋白(GFAP)阳性的星形胶质细胞及2',3'-环核苷酸-3'-磷酸二酯酶(CNP)阳性的少突胶质细胞,细胞免疫荧光及RT-PCR证实神经干细胞表达趋化因子受体CX3CR1.结论 新生大鼠海马神经干细胞表达趋化因子受体CX3CR1.为进一步研究其体内外迁移提供理论依据.     

恒河猴骨髓基质细胞向成熟神经细胞分化的实验研究

目的研究恒河猴骨髓基质细胞(BMSCs)在体外向神经细胞诱导分化过程中神经递质分泌和神经细胞抗原表型表达情况。方法无菌条件下密度梯度法分离猴BMSCs,在体外应用神经干细胞培养液进行体外培养和诱导分化,分阶段用高效液相色谱法检测培养基中单胺类生物活性物质的含量,免疫细胞化学方法检测神经细胞抗原表型。结果高效液相检测神经干细胞培养基未测到单胺类生物活性物质,而经体外培养增殖10d、14d、30d的细胞培养基可检测到去甲肾上腺素 (NE)和多巴胺(DA)。Asp方差分析显示:随着BMSCs培养天数的增加,培养基中所含的NE和DA尤显著性差异(P>0．05)。同期细胞免疫组化检测出酪氨酸羟化酶(TH)、巢蛋白抗体(nestin)、β-微管蛋白 (β-tublin)、胶质原性纤维酸性蛋白抗体(GFALP)和神经元特异烯醇化酶(NSE)抗原表达。结论恒河猴BMSCs能在体外增殖,在适宜条件下能分化成神经元样细胞,并合成、分泌NE和DA;部分细胞表达神经干细胞、成熟神经元、胶质细胞和DA能神经元的抗原表型,提示在一定条件下,诱导分化的 BMSCs经过神经干细胞阶段可向成熟神经组织细胞分化。     

PI3K/AKT/mTOR信号转导通路相关蛋白在脑胶质瘤中的表达及临床意义

目的研究PI3K/AKT/mTOR信号转导通路相关蛋白PI3K、mTOR、PTEN在脑胶质瘤中的表达及临床意义。方法采用免疫组化Envision二步法检测75例患者的脑胶质瘤标本(胶质瘤组,其中肿瘤分级Ⅱ级30例,Ⅳ级45例)及10名正常人(正常对照组)脑组织PI3K、mTOR、PTEN蛋白的表达,并对脑胶质瘤患者进行生存的相关分析。结果 PI3K、mTOR蛋白在胶质瘤组的阳性率显著高于正常对照组(均P<0.01),PTEN阳性率显著低于正常对照组(P<0.05)。Ⅱ级脑胶质瘤标本中PI3K、mTOR蛋白阳性率显著低于Ⅳ级(均P<0.01),PTEN阳性率显著高于Ⅳ级(P<0.05)。PTEN与PI3K、mTOR蛋白在胶质瘤中的表达分别呈负相关(r=-0.565,P=0.000;r=-0.322,P=0.005);PI3K与mTOR的表达呈正相关(r=0.456,P=0.000)。单因素分析显示,年龄、肿瘤级别、术后放化疗及PI3K蛋白表达影响患者术后的生存时间(r=-0.306,r=-0.422,r=0.392,r=-0.320;均P<0.05)。多因素回归分析显示,肿瘤级别、术后放化疗及PI3K蛋白表达为影响胶质瘤患者预后的独立因素(χ2=17.568,χ2=4.171,χ2=4.427;均P<0.05)。结论脑胶质瘤PI3K、mTOR的表达增高,PTEN的表达降低,并与肿瘤的恶性程度有关。肿瘤级别、术后放化疗及PI3K蛋白表达水平是影响胶质瘤患者预后的独立因素。     

法舒地尔和RhoA沉默对神经干细胞增殖的影响

背景：Rho及其相关分子在神经轴突生长、分化、延伸及突触形成中起重要作用,阻断和抑制RhoA/ROCK通路可促进神经干细胞的增殖与生长。 目的：观察Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默对大鼠神经干细胞增殖的影响。 方法：体外培养Wistar胎鼠神经干细胞,分6组干预：空白对照组,5,10,15,20 μmol/L 法舒地尔组,siRNA 沉默RhoA基因组。干预后第3天,采用RT-PCR,Western blot检测各组神经干细胞RhoA基因及蛋白的表达。应用MTT比色法观察神经干细胞增殖情况;采用流式细胞术测定神经干细胞周期分布的变化。 结果与结论：15,20 μmol/L 法舒地尔组、siRNA 沉默RhoA基因组神经干细胞RhoA基因及蛋白表达量较5,10 μmol/L 法舒地尔组、空白对照组明显降低(P < 0.05),细胞的生长速度较5,10 μmol/L 法舒地尔组、空白对照组明显增快(P < 0.05),细胞周期G0/G1期减少(P < 0.05),S期细胞数增多(P < 0.05)。当法舒地尔浓度增加到20 μmol/L时对细胞的作用并非随浓度的增加而增强,与15 μmol/L组的差异无显著性意义(P > 0.05)。15,20 μmol/L 法舒地尔组与siRNA 沉默RhoA基因组相比差异无显著性意义(P > 0.05)。说明Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默在体外均能促进神经干细胞增殖,法舒地尔最佳作用浓度为15 μmol/L。     

人胚胎干细胞神经分化过程中VEGF作用机制的研究

目的观察血管内皮生长因子(vascular endothelial growth factor,VEGF)对人胚胎干细胞分化为神经元的促进作用是否与Notch信号通路有关。方法人胚胎干细胞经拟胚体向神经元分化,并分为3组:A组为常规诱导组;B组为常规诱导+VEGF(10ng/ml)作用组;C组为常规诱导+γ-分泌酶抑制剂预处理+VEGF(10ng/ml)作用组。用免疫荧光法检测及计算各组不同阶段细胞阳性率,半定量RT-PCR观察各组神经干细胞Hes1 mRNA表达,MTT法检测3组神经干细胞增殖速率。结果免疫荧光法检测显示,B组产生神经干细胞的阳性率明显高于A、C两组,差异有统计学意义(P0.01),A、C两组之间无显著性差异(P0.05);B、C两组进一步分化为神经元的阳性率均明显高于A组,差异有统计学意义(P0.01),B、C两组之间无显著性差异(P0.05);半定量RT-PCR观察显示B组神经干细胞Hes1 mRNA表达明显高于A、C两组;MTT法检测与半定量RT-PCR结果相似。结论人胚胎干细胞体外分化过程中血管内皮生长因子通过激活Notch信号通路,促进神经干细胞增殖,而VEGF在神经干细胞向神经元分化中的作用与Notch通路无关。     

新生大鼠海马神经干细胞表达趋化因子受体CCR5的体外研究

目的探讨体外培养神经干细胞是否表达趋化因子受体CCR5。方法采用无血清方法分离、培养新生大鼠海马神经干细胞，细胞免疫荧光方法检测神经干细胞标志巢蛋白（nestin）表达及干细胞多向分化为神经元及胶质细胞的能力；后通过细胞免疫荧光及逆反转录酶一聚合酶链反应（RTPCR）方法检测神经干细胞表达趋化因子受体CCR5的情况。结果体外培养新生大鼠海马神经干细胞呈nestin阳性，可分化为神经丝蛋白200（NF200）阳性神经元、胶质纤维酸性蛋白（GFAP）阳性星形胶质细胞及2，3-环核苷酸磷酸二酯酶（CNP）阳性少突胶质细胞，CCR5细胞免疫荧光及RT-PCR证实神经干细胞表达趋化因子受体CCR5。结论新生大鼠海马神经干细胞表达趋化因子受体CCR5，为进一步研究其体内、外迁移提供理论依据。     

体外诱导骨髓基质干细胞向神经细胞分化及蛋白的差异表达***☆

背景：骨髓基质干细胞移植对损伤脊髓有一定修复作用，较神经干细胞移植更为理想，但疗效并不稳定，可能与其移植微环境有关。 目的：拟建立体外神经细胞微环境作用下骨髓基质干细胞的分化模型，观察其分化过程中蛋白表达的差异。 设计、时间及地点：蛋白水平的观察对照实验，于2005-07/2007-05在哈尔滨医科大学基础医学院神经生物实验室完成。 材料：Wistar成年大鼠及新生胎鼠。 方法：取新生Wistar胎鼠脊髓，以培养神经细胞。从成年Wistar大鼠骨髓中分离骨髓基质干细胞进行体外培养和增殖，应用红色荧光蛋白PKH26标记骨髓基质干细胞。骨髓基质干细胞、神经细胞单独培养组分别将骨髓基质干细胞、神经细胞单独培养，共培养组、分层联合培养组分别将标记的骨髓基质干细胞与神经细胞在体外共培养及在双层培养皿中联合培养。 主要观察指标：培养7 d后收集细胞分别进行神经特异性烯醇化酶和胶质纤维酸性蛋白免疫荧光检测。应用 SELDI-TOF-MS 技术筛选骨髓基质干细胞向神经细胞分化过程中变化明显的相关蛋白进行分析。 结果：骨髓基质干细胞与神经细胞共培养和双层联合培养7 d后，骨髓基质干细胞呈类似神经细胞形态。免疫荧光检测结果示，共培养组骨髓基质干细胞的神经特异性烯醇化酶和胶质纤维酸性蛋白阳性率明显高于分层联合培养组（P < 0.05），分层联合培养组明显高于单独培养对照组（P < 0.05）。骨髓基质干细胞在向神经细胞转化过程中有5种蛋白表达发生明显变化：在分层联合培养组TIP39_RAT和CALC_RAT表达增加，为原表达量的5.344和2.805倍；INSL6_RAT，PNOC_RAT和PCSK1_RAT表达下降，为原表达量的0.380，0.499和0.437倍。 结论：在体外神经细胞微环境作用下，骨髓基质干细胞与神经细胞在共培养和双层联合培养时均能诱导分化成神经细胞，接触培养比非接触培养分化率高。骨髓基质干细胞在向神经细胞转化过程中与5种蛋白TIP39_RAT，CALC_RAT，INSL6_RAT，PNOC_RAT和PCSK1_RAT密切相关。     

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 -