色素上皮源性因子与肿瘤

色素上皮源性因子 (Pigmentepithelium derivedfactor ,PEDF)是一种存在于正常组织中的可溶性糖蛋白。它具有营养和保护神经、抑制血管形成、促进肿瘤分化和凋亡等功能。PEDF是近年来国外研究较多的肿瘤生长和转移抑制因子之一 ,它的发现和其对肿瘤作用机制的阐明 ,对于人类恶性肿瘤的基础研究和临床应用具有重要的意义。     

牛膝多肽促大鼠海马神经元突起的生长

目的 研究牛膝多肽(ABPP)对体外培养的大鼠海马神经元突起生长的促进作用及其对生长相关蛋白-43 (GAP-43)和神经丝蛋白(NF-H)mRNA和蛋白表达的影响. 方法 以体外原代培养的胎鼠海马神经元为研究模型,通过四甲基偶氮唑盐(MTT)检测ABPP对海马神经元细胞活性的影响,通过免疫荧光细胞化学法,采用Image-Pro Express软件测量不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h,对海马神经元神经突起生长的影响;通过实时荧光定量PCR法定量分析不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药6h,对海马神经元GAP-43和NF-H基因表达的影响;采用免疫印迹法观察不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h后,对海马神经元GAP-43 和NF-H蛋白表达的影响.同时应用胞外信号调节激酶(ERK)特异性拮抗剂PD98059与ABPP共培养海马神经元,分析ABPP对海马神经元的作用与ERK通路的关系. 结果 ABPP可有效地促进海马神经元突起的生长,加药后24h浓度为1.0mg/L,作用最强;实时荧光定量RT-PCR、免疫荧光细胞化学法和免疫印迹检测的结果表明,ABPP能增加体外培养的海马神经元GAP-43和NF-H的mRNA和蛋白表达,PD98059可抑制该过程. 结论 ABPP能促进体外培养的海马神经元神经突起的生长,其作用与上调GAP-43和NF-H 基因的表达相关,并可能与ERK通路有关.     

大鼠海马神经元PKCε特异性shRNA对神经元突起生长的影响

目的 构建shRNA特异性干扰海马神经元内PKCε,研究PKCε干扰后海马神经元突起长度的变化.方法 设计并构建PKCε的小分子干扰shRNA载体.分别在293T细胞中共转染PKCε和各干扰片段,Western blot验证干扰效果.将有干扰作用的载体转染海马神经元,用细胞免疫荧光法验证干扰内源PKCε的效果.激光共聚...     

RNA干扰CRMP 5抑制大鼠海马神经元突起生长

目的探讨CRMP5对大鼠海马神经元突起生长的影响。方法将带FAM标记的CRMP5的特异性干扰片段及阴性对照转染培养成熟的海马神经元,用免疫荧光的方法验证干扰片段对神经元内源性CRMP5的干扰效果,并利用共聚焦显微镜观察神经元突起以及侧枝的形成。结果携带FAM的si RNA可以成功的进入细胞,分布于神经元的胞体以及树突;免疫荧光证实CRMP5 si RNA可以有效的沉默CRMP5蛋白的表达;沉默CRMP5基因表达后的海马神经元突起短小,而且缺少分支,而对照细胞突起长,分支多;定量分析显示,导入CRMP5 si RNA的细胞突起的长度较对照细胞缩短,差异显著(P0.05);突起的数目比较,一级突起数目无显著差异,而二级及其以上突起的数目明显减少,差异显著(P0.05)。结论沉默CRMP5可抑制海马神经元突起的生长和侧枝形成。     

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。     

大白鼠海马神经毯内神经胶质突起与神经元突起间关系的超微结构…

用电镜技术观察了大白鼠海马神经毯内的神经胶质突起与神经元突起的超微结构。本文论述了星状胶质细胞突起与神经元的树突、树突侧棘和轴突膜间联系的超微形态特点，发现星状胶质细胞突起广泛分布于神经毯内，与神经元突起间形成膜的紧密并列关系，但不形成突触，也未见二膜间并列处有膜的增厚和突触小泡聚集的现象。星状胶质细胞突起上的棘状突起的超微结构特征不同于树突的侧棘，无棘器的构造。产次发现有的神经胶质棘状突起上有小     

睫状神经营养因子对应激引起海马CA3神经元损害的作用

目的;探讨睫状神经营养因子对应激引起海马ＣＡ３区神经元损害的作用。方法：采用Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法和常规透射电镜技术,观察急性和慢性足底电击大鼠的海马ＣＡ３区神经元开矿的变化,及双侧海马注射睫状神经营养因子对其影响。结果;急性应激大鼠海马神经元形态无明显变化;慢性应激大鼠海马神经元出现明显的损伤性形态变化;睫状神经营养因子对正常大鼠和急性应激大鼠的海马神经元     

短暂性缺氧后海马神经元神经源性分化因子表达增高

目的 观察短暂性缺氧后神经源性分化因子(NeuroD)表达量的变化,探讨其在神经系统再生中的可能作用.方法 体外培养海马神经元,经神经元特异性烯醇化酶(NSE)免疫组织化学方法、尼氏体染色法鉴定.将培养5d的神经元置于三气培养箱(37℃、94%N2、5%CO2、1%O2)内缺氧培养.RT-PCR检测缺氧3h和6h后神经...     

GDNF促进大鼠背根神经元的存活和突起生长

探讨胶质细胞源性神经营养因子(GDNF)对正常胚胎大鼠背根神经节(DRGn)的存活及突起生长的作用。本实验采用神经组织原代分离培养的方法建立体外胚胎大鼠背根神经节单细胞培养体系，从细胞形态学及应用MTT。法观察1μg／L、10μg／L、50μg／L和100μg／L GDNF对体外培养的正常感觉神经元生长的影响。结果表明：GDNF能明显促进体外培养的正常大鼠背根神经节感觉神经元的存活及突起生长。提示GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

氧环境对血管内皮生长因子、色素上皮衍生因子的影响及其与血管发育的关系

目的：研究视网膜发育过程中血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)所起的调节作用及其机制。方法：C57BL/6J 小鼠从出生后7～12 d 饲养于75%氧环境,分别用抗 VEGF、抗 PEDF 和抗 CD31抗体作免疫组织化学标记视网膜切片。结果：持续处于高氧环境时,VEGF 表达处于低水平而 PEDF 快速上升,血管生长减缓。高氧处理5 d 后回到普通环境,VEGF 的表达迅速上升而 PEDF 却逐渐减少,出现了一过性的血管快速增长现象。结论：氧含量变化可调节 VEGF 和 PEDF 表达,参与视网膜血管发育。     

靶向性抗癌蛋白色素上皮衍生因子在肿瘤中的研究进展

在肿瘤靶向治疗研究中,抗血管生成治疗是近十年来的一个研究热点.目前已有许多血管生成抑制剂应用于临床试验中.色素上皮衍生因子(PEDF)是一个能有效抑制新生血管形成的蛋白,与血管内皮生长因子(VEGF)等共同调控新生血管的发生过程.PEDF表达和抑制肿瘤生长、减少转移、良好的预后相关,对PEDF的深入研究有助于发挥其在肿瘤治疗中的作用.     

Pigment epithelium derived factor (PEDF) is neuroprotective in two in vitro models of Parkinson's disease

Transplantation of retinal pigment epithelial (RPE) cells in the basal ganglia has been proposed as a novel cell-based therapy for Parkinson's disease (PD), by providing a constant source of dopamine replacement via the melanin synthetic pathway enzyme tyrosinase. We have demonstrated previously that human RPE cells also produce a neurotrophic effect on primary cultures of rat striata mesencephalic (dopaminergic) neurons and showed that pigment epithelium derived factor (PEDF) accounted for a major portion of the neurotrophic effect. We now have also begun studies that demonstrate that the neurotrophic effect of PEDF corresponds to neuroprotection against toxins used to produce experimental PD. This was shown in (1) rotenone and (2) 6-hydroxydopamine (6-OHDA) in vitro models. The toxins were added at day 10 in culture, PEDF was added 1 h prior. The cultures were fixed and analyzed after tyrosine hydroxylase (TH) immunocytochemical staining. Cell count of TH+ neurons clearly shows the neuroprotective potential of PEDF in both neurotoxin models. The neurotoxic effect of rotenone (25 nM) on dopaminergic neurons is reversed by addition of PEDF. At a concentration of 1 ng/ml PEDF the neurotoxic effect of rotenone is completely counteracted. PEDF (1 ng/ml) has also a neuroprotective effect in the 6-OHDA midbrain in vitro model. The effect is most pronounced at concentrations of 25 μM and 50 μM 6-OHDA. We conclude that the neurotrophic factor PEDF, produced from RPE cells, can improve neuronal survival in models of PD, and plan to test if this effect can be observed using in vivo models of PD following RPE transplantation.     

糖尿病大鼠视网膜色素上皮细胞超微结构

目的 :探讨糖尿病大鼠视网膜色素上皮细胞超微结构病变 ,为糖尿病视功能障碍提供有关的行态学依据。方法 :选择清洁级SD大鼠随机分成正常对照组、糖尿病 1月、3月和 6月。腹腔内注射链脲佐菌素 (STZ)诱导大鼠糖尿病 ,每组 1 2只 ,取视网膜制备超薄切片 ,透射电镜观察。处死前每月测体重、血糖 1次。结果 :糖网病大鼠视网膜色素上皮细胞的微绒毛和基底部的质膜内褶随血糖的增高病情加重而逐渐稀疏、低矮直至脱落、低平。胞质内细胞器以线粒体的脱嵴、水肿和内质网的扩张为主要损害 ,而相邻色素上皮细胞之间的连接复合体始终存在。结论 :糖网病大鼠视网膜色素上皮细胞的损害主要表现于微绒毛、质膜内褶及细胞器的损害 ,并无脉络膜与视网膜之间屏障功能的损害。本研究为糖网病的临床研究提供形态学依据     

Differential effects of phencyclidine upon hippocampal complex-spike and theta neurons

The effects of local application of phencyclidine (PCP) upon hippocampal CA1 neurons were investigated in urethane-anesthetized rats. Hippocampal neurons were classified on the basis of extracellularly recorded action potential duration as either complex-spike or theta cells prior to PCP administration. PCP depressed spontaneous firing of 46 of 48 complex-spike cells, but excited 12 of 13 theta neurons. This result demonstrates that hippocampal complex-spike and theta neurons may be differentiated of theta neurons were greatly attenuated or absent in rats pretreated with DSP4, a neurotoxin which selectively destroys noradrenergic pathways. This latter finding lends additional support to the hypothesis that the effects of locally applied PCP are mediated via noradrenergic mechanisms.     

Mammalian neurons in dissociated cultures form clusters in the presence of retinal pigment epithelium

Summary The objective of this study was to investigate the cellular processes involved in the formation of the cytoarchitectonics of the retina. Neurons derived from the retina, spinal cord, cerebral cortex and hippocampus were grown in dissociated monolayer tissue culture using standard techniques. The cultures of retina were unique in that the neurons actively formed into itcell clusters. On the other hand, cultures of neurons from the other regions of the CNS grew without forming any obvious histotypical pattern. Cell clusters consisted of an apparent monolayer of neurons above a population of flat cells and clusters were observed in retinal cultures derived from all species studied (mouse, cat and guinea pig). Each cluster was surrounded by whorls of fibroblasts; astrocytes (GFAP-positive cells) were often closely associated with clusters. Formation of clusters appeared to depend strongly upon the presence of cells derived from the retinal pigment epithelium (RPE) because the ability of retinal cells to form clusters was markedly impaired when the RPE was omitted from the cultures. Interestingly, monolayer cultures of neurons from other regions of the CNS could be induced to form clusters, but only when cells of the RPE layer were included at the time of plating. In cultures grown without the RPE layer, clusters did not form when media taken from cultures expressing clusters was used, indicating that the formation of clusters was not caused by a mediabourne factor. On the other hand, clusters did form when neurons without RPE were grown on feeder plates in which clusters had previously been expressed and the neurons subsequently killed by prolonged culturing or by treatment with kainic acid. Hence, physical contact between neurons and cells derived from the RPE appears critical for the formation of clusters. Our results suggest that the cellular processes underlying the formation of clusters may reflect those in the development of the retina in vivo. Thus, cluster formation may be a useful model for investigating the initial stages in the development of retinal cytoarchitecture.     

坍塌反应调节蛋白5促进海马神经元突起生长

目的 探讨坍塌反应调节蛋白5(CRMP5)对神经元突起生长的作用。方法 构建CRMP5真核表达载体,采用基因转染、实时定量PCR和免疫印迹技术评估CRMP5基因表达;以空载体为对照组,设3个复孔,用时差成像技术和突起提取技术观察和检测原代培养海马神经元突起的生长。结果 成功构建携带增强型绿色荧光蛋白（EGFP）标签蛋白的CRMP5的真核表达载体。脂质体转染技术可成功把CRMP5基因导入细胞,转染的细胞CRMP5表达高于空载体对照组;CRMP5蛋白表达于神经元的胞体和突起,尤其是胞体、突起起始处和突起末端高表达。过表达CRMP5可明显促进突起生长,主要表现为突起的生长,并形成丰富的侧枝;定量结果显示,CRMP5过表达的细胞突起的长度逐渐延长,而且较空载体转染细胞增多,差异显著(P<0.01)。导入CRMP5的细胞突起提取液的吸光度较对照细胞明显升高(P.<0.01)。 结论 CRMP5能促进神经元突起及其分支的生长。     

视网膜色素上皮腺瘤的病理形态学观察

目的　探讨视网膜色素上皮腺瘤临床及形态学特征 ,为其诊断及鉴别提供依据。方法　常规石蜡切片HE染色、组织化学PAS和VG染色、透射电镜及用SP法做免疫组化S 10 0、Cytokeratin和Vimentin检测。结果　该瘤细胞呈椭圆形、立方形 ,部分瘤细胞排列成腺管状 ,瘤细胞团周围见均匀红染的条状物质 ;上述条状物质PAS染色阳性 ,VG染色大部分呈黄色 ,少量呈红色 ;透射电镜显示细胞的紧密连接 ;免疫组化S 10 0和Cytokeratin阳性、vimentin阴性。结论　视网膜色素上皮腺瘤的超微结构及免疫组化特征均同视网膜色素上皮相似 ,可通过形态学检查确诊。     

早期人胚胎视网膜色素上皮细胞的体外培养及生物学特性

目的探索早期人胚视网膜色素上皮(RPE)细胞体外培养以获得高纯度的RPE细胞的方法,并进一步研究其生物学特性。方法取意外流产4h以内人胚(6～10周)眼球,通过混合酶消化法获取RPE细胞。用F12培养液体外培养并作细胞形态学、S-100和Keratin免疫组织化学鉴定;通过Trizol一步法提取总RNA,逆转录聚合酶反应(RT-PCR)分析体外培养RPE细胞酪氨酸酶基因的表达。结果用F12培养液可获得纯度高、活性好的人胚RPE细胞;体外培养的RPE细胞不表达酪氨酸酶基因。结论早期人胚RPE细胞具有在体RPE细胞的部分特征;体外培养的RPE细胞未检测到酪氨酸酶RNA表达,故随着细胞分裂增殖,色素颗粒逐渐减少,提示体内外RPE细胞在功能上存在某些差异。