E-钙粘素和β-连环素在脑膜瘤中的表达及意义

Objective To analyze the expressions of E-cadherin and β-catenin in meningioma by immunohistochemistry for further understanding of biological behaviors of meningiomas. Methods The specimens included in this study were collected form 49 meningioma cases. EnVision was used in immunohistochemieal staining. The results were graded depending on the positive rate and intensity of the immunoreactivity. E-cadherin and β-catenin in meningiomas were analyzed in relationship with WHO2000 grading and invasion. Results The positive rates of E-adhesion in meningioma WHO Ⅰ,Ⅱ,Ⅲ were 92.69%, 33.33% and 0, respectively (P<0.05). The positive rates of β-catenin in meningioma WHO Ⅰ,Ⅱ,Ⅲ were 82.93%, 33.33% and 20.00%, respectively (P<0.05). The positive rate of E-adhesion in meningiomas without invasion (94.12%) was higher than that in ones with invasion (46.67%), and the difference was of statistical significance (P<0.05). The difference in positive rate of β-catenin was statically significant between meningiomas without invasion (88.24%) and the ones with invasion (33.33%, P<0.05). Conclusions The levels of E-adhesion and β-catenin are in close correlations with the WHO2000 grading of meningioma. In the atypical or anaplastic meningiomas, the expressions of E-adhesion and β-catenin are lower significantly. The levels of E-adhesion and β-catenin are also in close correlations with the aggressiveness of meningioma. The lower the expressions of E-adhesion and β-catenin, the more invasive meningioma will possibly be.     

锂剂和氯胺酮对N2a细胞mTOR及GSK-3β的影响

目的 观察锂剂和氯胺酮分别对N2a细胞中西罗莫司靶蛋白(mTOR)及糖原合酶激酶-3β( GSK-3β)的影响.方法 将N2a细胞随机分为3组(n=6),分别加入DMEM培养液(C组)、3μmol/L氯化锂(L组),1μmol/L氯胺酮(K组),孵育2d后采用Western Blotting分别检测mTOR和GSK- 3β表达.结果 与C组相比,L组和K组mTOR表达显著上调,GSK- 3β表达显著下调(P＜0.05).结论 锂剂和氯胺酮可上调mTOR,下调GSK- 3β.     

糖原合成酶激酶-3β在帕金森模型小鼠中的改变特点

目的 观察糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)在帕金森病(Parkinson's disesse,PD)的动物模型中变化的规律.方法 利用1-甲基-4苯基-1,2,3,6-四氢吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)建立经典的PD小鼠动物模型.采用免疫组化、Western Blot方法 ,观察酪氨酸羟化酶(tyrosine hydfoxylase, TH)和GSK-3β在该模型中的表达变化规律.结果 TH免疫染色:MPTP处理后小鼠中脑TH阳性反应明显减少.Western Blot:在腹侧中脑,连续MPTP处理后总GSK-3β和磷酸化的GSK-3β(pllosphorylated GSK-3β,p-GSK-3β)均明显升高,然而p-GSK-3β/总GSK-3β比值降低,所以非磷酸化的GSK-3β是增高的.免疫组织化学:MPTP处理后GSK-3β在腹侧中脑的表达明显增强.结论 在MPTP处理的小鼠PD模型中,腹侧中脑的多巴胺能神经元减少,而GSK-3β在该区域特异性的增高,GSK-3β可能参与了中脑神经元的损伤过程.     

14-3-3蛋白对其他功能蛋白亚细胞定位的调控作用

14-3-3蛋白是所有真核细胞均表达的一组相对分子质量为(28～33)×103的酸性蛋白,该蛋白家族成员的氨基酸序列及功能等均具有高度保守性.已在哺乳动物的细胞中发现分别由不同基因编码的7种14-3-3蛋白亚型(β、γ、ε、σ、ξ、τ、η),它们主要存在于细胞质中,可自行组装成纯合型或杂合型二聚体.     

创伤性颅脑伤后小鼠皮层和海马Wnt3a和β-Catenin的表达变化

目的探讨创伤性脑损伤后小鼠皮层和海马Wnt3a和β-Catenin的表达及其意义。方法在小鼠创伤性脑损伤模型基础上,采用免疫印迹法和免疫组织化学法检测创伤性脑损伤后Wnt3a和β-Catenin的蛋白变化。结果 Wnt3a和β-Catenin在对照组的脑组织中表达较低。创伤性脑损伤后1 h,皮层和海马区β-Catenin和Wnt3 a的含量开始增加。与对照组相比,伤后6h和第7天,β-Catenin和Wnt3a蛋白表达显著增加(P0.05)。免疫荧光染色结果显示,脑损伤24 h后,β-Catenin和Wnt3a在小鼠皮层和海马表达量均增加。结论小鼠创伤性脑损伤后早期和恢复期,Wnt3a和β-Catenin的蛋白水平在皮层和海马显著增高,这提示Wnt3a和β-Catenin可能在创伤性脑损伤的病理生理机制中发挥着重要的作用。     

Wnt3a促进大鼠骨髓间充质干细胞向神经元样细胞的分化

背景：Wnt信号通路是细胞增殖分化的关键调控环节,但与骨髓间充质干细胞神经分化的联系并不十分明确。 目的：寻找促进骨髓间充质干细胞向神经元样细胞分化的Wnt信号分子。 方法：首先体外分离培养大鼠骨髓间充质干细胞并传代,行形态学观察,并以流式细胞学方法检测细胞表型CD44,CD9,CD34和CD45。采用碱性成纤维细胞生长因子分别联合Wnt3a或Wnt5a的方案诱导分化,应用免疫组化和反转录-聚合酶链反应方法比较Wnt3a和Wnt5a对骨髓间充质干细胞向神经元样细胞分化的影响。 结果与结论：骨髓间充质干细胞为长梭形,CD9,CD44高表达,CD34,CD45低表达。Wnt3a诱导组的巢蛋白和神经元特异烯醇化酶呈阳性,而胶质纤维酸性蛋白无明显表达,诱导后细胞的活力良好。Wnt5a诱导组巢蛋白呈弱阳性表达,而神经元特异烯醇化酶及胶质纤维酸性蛋白阴性。反转录-聚合酶链反应结果显示,Wnt3a诱导组巢蛋白在诱导前后均有表达,神经元特异烯醇化酶在诱导后5 d可见明显的扩增条带,10 d后更加明显。胶质纤维酸性蛋白在诱导10 d后出现较弱的扩增条带。Wnt5a组、对照组骨髓间充质干细胞在诱导后10 d巢蛋白有微弱表达,神经元特异烯醇化酶和胶质纤维酸性蛋白几乎无表达。提示Wnt3a分子能够促进体外培养的骨髓间充质干细胞向神经元样细胞分化。     

14-3-3蛋白在PC12细胞的分布及MPP+对其表达的影响

目的探讨14-3-3蛋白在PC12细胞中的分布及其与MPP 诱导PC12细胞死亡的关系。方法用免疫荧光染色检测14-3-3蛋白的分布,并分别用免疫印迹技术与MTT法检测MPP ,对14-3-3蛋白表达及细胞活力的影响。结果PC12细胞中β、γ、ε、ζ、η和θ亚型免疫反应阳性。14-3-3蛋白表达随着MPP 处理的时间延长呈下降趋势,在6h变化不明显,12h显著下降(P<0.05),到48h后14-3-3蛋白的减少极其显著(P<0.01)。PC12细胞的存活率也呈现同样的变化趋势。结论MPP 引起PC12细胞的死亡可能与细胞内14-3-3蛋白含量的下降有关。     

Wnt/β-连环蛋白信号通路参与调节糖皮质激素诱导的阿尔茨海默病样病变

目的 探讨Wnt/β-连环蛋白(catenin)信号通路在糖皮质激素(glucocorticoids,GC)诱导的阿尔茨海默病样病变中的信号调节机制.方法 地塞米松(dexamethasone,DEX)分别处理转染人tau441的人胚肾293细胞(HEK293/tau)和野生型的人胚肾293细胞(HEK293/wt),采用细胞计数试剂盒( CCK-8)法检测DEX对细胞活力的影响,Western blot研究两组细胞tau蛋白磷酸化(p-T205,Tau-1)、β-catenin、p-β-catenin、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)及其上游激酶糖原合成激酶-3β(GSK-3β)、ps9-GSK-3β水平的变化,并加用GSK-3β的抑制剂氯化锂(LiCI),观察其对相关蛋白相应的逆转作用.结果 CCK-8细胞活力检测结果显示,1μmol/L DEX处理细胞48h,HEK293/wt组细胞活力(存活率)下降到95.5％±3.2％,HEK293/tau组下降到77.8％±4.4％,两者差异有统计学意义(t =6.60,P＜0.05);Western blot结果显示,1μmoL/L DEX处理两组细胞48 h,使得HEK293/tau细胞ps9-GSK3β、Tau -I、β-catenin、Bcl-2水平分别下降到对照组的47.8％±10.4％、53.9％±11.7％、50.9％±7.6％、48.4％±6.5％,差异均有统计学意义(t=7.01、3.86、7.09、7.30,均P＜0.05),而p-T205、p-β-catenin相对磷酸化水平分别增加到对照组的180.5％±22.2％、201.3％±27.6％,差异均有统计学意义(t=5.51、5.27,均P＜0.05);LiCI可以相应逆转上述改变.结论 在人tau存在的前提下,GC通过抑制Wnt/β-catenin信号转导通路,促进了HEK293/tau细胞的阿尔茨海默病样病变.     

灯盏花素通过Wnt/β-catenin通路对阿尔茨海默病大鼠模型Aβ跨血脑屏障的转运清除机制研究

目的 探讨灯盏花素（Breviscapine,BRE）对阿尔茨海默病（Alzheimer disease,AD）大鼠模型淀粉样蛋白β（Amyloid β,Aβ）跨血脑屏障（blood brain barrier,BBB）的转运清除的影响及其可能作用机制。 方法 随机选择15只Wistar大鼠作为空白组,剩余大鼠于侧脑室内注射淀粉样蛋白β25-35（Aβ25-35）以诱导AD大鼠模型;后随机分为模型组、BRE低（50 mg·kg-1·d-1）、高（100 mg·kg-1·d-1）剂量组及多奈哌齐组（1 mg·kg-1·d-1）,每组各15只,连续灌服给药21 d;Morris水迷宫行为测试检测大鼠空间学习记忆能力;海马组织行尼氏、刚果红染色;酶联免疫吸附试验（Enzyme linked immunosorbent assay,ELISA）法检测海马组织中Aβ42、肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）、白细胞介素1β（Interleukin 1β,IL-1β）、丙二醛（Malondialdehyde,MDA）、超氧化物歧化酶（Superoxide dismutase,SOD）水平;Western blot法检测海马Wnt3a/β-连环蛋白（β-catenin）通路因子及Aβ相关转运体蛋白表达水平。 结果 与空白组比较,模型组大鼠逃避潜伏期延长,穿台次数减少,海马CA3区存活神经元数减少,Aβ斑块状严重沉积,Aβ42,TNF-α,IL-1β,MDA水平升高,SOD水平、Wnt3a,β-catenin、低密度脂蛋白受体相关蛋白1（Low density lipoprotein receptor-associated protein 1,LRP-1）及P糖蛋白（P glycoprotein,P-gp）相对表达水平降低,磷酸化糖原合酶激酶3β（Phosphorylated GSK3 β,p-GSK3β）、轴抑制蛋白2（Axis inhibition protein 2,Axin2）相对表达水平升高（P<0.05）;与模型组比较,BRE低、高剂量组及多奈哌齐组大鼠逃避潜伏期缩短,穿台次数增加,海马CA3区存活神经元数增加,Aβ斑块状沉积减轻,Aβ42,TNF-α,IL-1β,MDA水平降低,SOD水平、Wnt3a,β-catenin,LRP-1及P-gp相对表达水平升高,p-GSK3β,Axin2相对表达水平降低（P<0.05）;低、高剂量BRE及多奈哌齐作用效果逐渐增强（P<0.05）。 结论 BRE可促进AD大鼠模型Aβ跨BBB转运清除,其作用机制可能与激活Wnt/β-catenin通路有关。     

亲环素A对Aβ25-35诱导PC12细胞凋亡的影响及机制研究

目的 探讨亲环素A(CyPA)对Aβ25-35诱导的PC12细胞凋亡影响及可能的作用机制.方法 将PC12细胞分为正常对照组(0 μmol/L Aβ25-35)、Aβ25-35诱导组(10 μmol/L Aβ25-35)和药物保护组(0.1、1、10、100 nmol/L CyPA+10 μmoi/Lβ25-35),药物保护组采用相应浓度CyPA预处理PC12细胞30 min,再加入Aβ25-35继续培养.MTT法分析细胞存活率,Hoechst33258染色法检测细胞凋亡,RT-PCR检测Bcl-2和Bax mRNA表达,Western blotting检测Bcl-2和Bax蛋白表达.结果 1、10和100 nmol/L CyPA可提高细胞的存活率,减少Aβ25-35引起的细胞凋亡,增加Bcl-2mRNA表达以及Bcl-2蛋白表达,降低Bax mRNA表达以及Bax蛋白表达,与Aβ25-35诱导组比较差异有统计学意义(P<0.05),且CyPA剂量越高效果越明显.结论 CyPA可剂量依赖性对抗Aβ25-35对PC12细胞的毒性作用,减少细胞凋亡,其机制与上调抗凋亡基因Bcl-2表达和下调凋亡基因Bax表达有关.

Abstract：

Objective To explore the effect of cyclophilin A (CyPA) on apoptosis of PC 12 cells induced by Aβ25-35 and its potential mechanism. Methods PC 12 cells were divided into normal control group (0 μmol/L Aβ25-35), Aβ25-35 inducement group (10 μmol/L Aβ25-35) and drug protection groups (0.1, 1,10 and 100 nmol/L CyPA+10 μmol/L Aβ25-35). Cells in the drug protection groups were pretreated by CyPA of different concentrations for 30 min, and then co-cultured with Aβ25-35 We evaluated the survival rate of PC12 cells with MTT assay, analyzed the apoptosis of PC12 cells with Hoechst33258 staining, and detected the mRNA expressions of Bcl-2 and Bax with PT-PCR and the protein levels of Bcl-2 and Bax with Western blotting. Results Cells pretreated wth CyPA of 1, 10 and 100 nmol/L enjoyed an obvious elevation of survival rate of PC 12 cells, a significant reduction of apoptosis induced by Aβ25-35,an obvious increase of mRNA expression of Bcl-2 and protein level of Bcl-2, and a statistical decrease of mRNA expression of Bax and protein level of Bax as compared with those cells of the Aβ25-35 inducement group (P<0.05);and these effects were dose-dependent. Conclusion CyPA could resist the toxic role of Aβ25-35 on PC 12 cells and reduce the apoptosis in a dose-dependent manner by up-regulation of anti-apoptosis gene Bcl-2 and down-regulation of apoptosis gene Bax.     

Annexin-1 (lipocortin-1)-immunoreactivity in the folliculo-stellate cells of rat anterior pituitary: the effect of adrenalectomy and corticosterone treatment on its subcellular distribution

In the pituitary gland, annexin-1 (lipocortin-1) located in folliculo-stellate (FS) cells has been advocated as one of the candidates for paracrine agents produced by FS cells that modulate the release of pituitary hormones. However, the expression and distribution pattern of annexin-1 in FS cells under different circulating corticosteroid conditions has not been examined. Thus, by means of pre-embedding immunoelectron microscopy, we investigated the expression of annexin-1 in FS cells under different corticosteroid conditions. Annexin-1-immunoreactivity was observed in the cytoplasm; especially intense immunoreactivity was detected in the follicle surface of FS cells under control conditions. After adrenalectomy, annexin-1-immunoreactivity almost disappeared, but the immunoreactivity recovered with corticosterone replacement. The expression of glucocorticoid receptor immunoreactivity in the nucleus of FS cells also showed a similar pattern to annexin-1 associated with the changes in the corticosteroid conditions. However, S-100 immunoreactivity, a marker for FS cells, was not changed whatever the corticosteroid conditions. These results confirm that glucocorticoids regulate the annexin-1 expression and demonstrate the translocation of annexin-1 from intracellular to pericellular sites in the FS cells of the rat anterior pituitary gland.     

Rapid antidepressive-like activity of specific glycogen synthase kinase-3 inhibitor and its effect on beta-catenin in mouse hippocampus.

BACKGROUND: Inhibition of glycogen synthase kinase-3 (GSK-3) is thought to be a key feature in the therapeutic mechanism of several mood stabilizers; however, the role of GSK-3 in depressive behavior has not been determined. In these studies, we evaluated the antidepressive effect of L803-mts, a novel GSK-3 peptide inhibitor, in an animal model of depression, the mouse forced swimming test (FST). METHODS: Animals were intracerebroventricularly injected with L803-mts or with respective control peptide (cp) 1 hour, 3 hours, or 12 hours before their subjection to FST. RESULTS: Animals administered L803-mts showed reduced duration of immobility at all three time points tested, as compared with cp-treated animals. Expression levels of beta-catenin, the endogenous substrate of GSK-3, increased in the hippocampus of L803-mts-treated animals by 20%-50%, as compared with cp-treated animals. CONCLUSIONS: Our studies show, for the first time, that in-vivo inhibition of GSK-3 produces antidepressive-like behavior and suggest the potential of GSK-3 inhibitors as antidepressants.     

人重组NT-3基因体外转染胚胎干细胞及其表达

目的 探讨神经营养素-3(NT-3)基因体外转染小鼠胚胎干细胞(ES)的可能性及其表达情况.方法 用脂质体介导的方法,将重组真核表达载体PCDNA-3.1(+).NT-3瞬时转染ES.用细胞免疫组化及RT-PCR检测转染细胞NT-3蛋白及mRNA表达.ELISA检测细胞分泌上清液中NT-3蛋白表达.结果 细胞免疫组化结果显示,转染NT-3基因的ES细胞胞浆呈红色染色;RT-PCR得到200 bp的基因片段;ELISA检测细胞分泌上清液中NT-3蛋白表达呈阳性,与对照组有显著差别(P＜0.05).结论 重组真核表达载体PCDNA-3.1(+).NT-3可成功转染ES,获得稳定表达NT-3的ES细胞株.     

The effect of the acetylcholine transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine in mouse brain

The effect of the acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine was studied in mouse forebrain. Results indicated that AH5183 reduced the amount of ACh released from mouse forebrain minces by high K+ and veratridine over the identical concentration range as it inhibits the active transport of ACh into synaptic vesicles isolated from the electric organ of Torpedo. However, AH5183 did not block the K+- or veratridine-induced reduction of cytoplasmic (S3) ACh. Also, it did not block the loss of vesicular (P3) ACh caused by these depolarizing agents. It did, however, cause a disappearance of nerve ending ACh which was partially matched by a selective gain in the choline content of the P3 fraction. When minces of mouse forebrain were pretreated in high K+ to deplete the S3 and P3 fractions of their ACh content and then subsequently incubated in normal Krebs with [14C]choline, AH5183, at a concentration which reduces ACh release by 50%, did not affect the repletion of P3 stores with newly synthesized [14C]ACh. At somewhat higher concentrations, however, AH5183 reduced the amount of [14C]ACh in the P3 fraction without affecting the amount of [14C]ACh in the S3 fraction. At these concentrations it did not inhibit extracellular choline transport or ChAT activity. These results suggest that AH5183 may reduce the amount of ACh released from central cholinergic nerve terminals in response to depolarization through a combination of effects: (1) it may facilitate the breakdown or loss of ACh stored in the vesicular fraction; (2) it may also block the transport of newly synthesized ACh into the vesicular fraction.     

Differential distribution of the members of the dystrophin glycoprotein complex in mouse retina: effect of the mdx(3Cv) mutation

Dystrophin glycoprotein complex (DGC) assembly and function require mediation by dystrophin in skeletal muscle. The existence of such complexes and the correlation with DMD phenotypes are not yet established in the central nervous system. Here we have studied the expression of DMD gene mRNAs and proteins in retina from C57BL/6 and mdx(3Cv) mouse strains. Then we have comparatively investigated the localization of dystrophin and dystrophin-associated proteins (DAPs) in both strains to analyze the repercussion of the mdx(3Cv) mutation on the retinal distributions of alpha/beta-dystroglycan, alpha1-syntrophin, alpha-dystrobrevin, and delta/gamma-sarcoglycan. Results showed that DMD gene product deficiency affects the expression of dystroglycan assembly exclusively at the outer plexiform layer without an apparent effect on the other DAPs. We conclude that the localization of members of the DGC could be independent of the presence of the DMD gene products and/or utrophin.     

Transfection and stable transformation of adult mouse Schwann cells with SV-40 large T antigen gene

Cultured Schwann cells derived from adult mouse dorsal root ganglia and peripheral nerves were transfected with a plasmid containing SV-40 large T antigen gene, and 25 colonies of stable transformants were obtained, one of which was expanded and recloned. This transfected cell line, designated MS1, expressed SV-40 large T antigen and showed continuous cell growth with a doubling time of 27 hours. The MS1 cells had distinct Schwann cell phenotypes such as S-100 protein, laminin, 2',3'-cyclic nucleotide 3'-phosphodiesterase and P0 protein, as shown by immunofluorescence microscopy. When MS1 cells were exposed to dibutyryl cyclic adenosine-3',5'-monophosphate (dbc AMP), they extended long bipolar processes two- to ten-fold longer than those of untreated MS1 cells and frequently formed whorl-like alignments similar to palisade formations or organoid patterns observed in human Schwannomas and neurofibromas. These results suggest that transformed Schwann cells can be a useful model for analyzing regulatory mechanisms of Schwann cells, neuron-Schwann cell interactions and experimental Schwann cell neoplasms in vitro.     

Vasoactive intestinal peptide (VIP): Brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats

We have studied the regional and subcellular distribution of vasoactive intestinal peptide (VIP) in the brain of adult male rat, using a specific radioimmunoassay. Selective deafferentation of the mediobasal hypothalamus (MBH) was also performed in order to investigate the origin of hypothalamic VIP. The highest concentrations of VIP were found in the neocortex, namely the occipital region. The brain stem, the posterior hypothalamus, and the pineal gland contained low amounts of the peptide. VIP was not detectable in the cerebellum and the neurohypophysis. After fractionation, most of the VIP was recovered from the crude mitochondrial fraction of the hypothalamus as well as the parietal cortex. However, a non-negligible portion of the activity was also found in the supernatant suggesting that the peptide is mainly located in nerve endings but also present in neuronal cell bodies and/or axons. Two weeks after complete deafferentation of the MBH, VIP concentrations of the caudal MBH (including the infundibular sulcus, the stalk, part of the ventromedial nucleus and premamillary structures) were decreased by 40%. In contrast, no change in VIP levels were observed in the rostral MBH, organum vasculosum of the lamina terminalis (OVLT), and cortex. This suggests that hypothalamic nerve endings containing the peptide derive from neuronal cell bodies located both outside and within the MBH.     

The subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABA(A) receptors.

GABA(A) receptors the major sites of fast synaptic inhibition in the brain are composed predominately of alpha, beta, and gamma2 subunits. The receptor gamma2 subunit interacts with a 17-kDa microtubule associated protein GABARAP, but the significance of this interaction remains unknown. Here we demonstrate that GABARAP, which immunoprecipitates with GABA(A) receptors, is not found at significant levels within inhibitory synapses, but is enriched within the Golgi apparatus and postsynaptic cisternae. We also demonstrate that GABARAP binds directly to N-ethylmaleimide-sensitive factor (NSF), a protein critical for intracellular membrane trafficking events. NSF and GABARAP complexes could be detected in neurons and these two proteins also colocalize within intracellular membrane compartments. Together our observations suggest that GABARAP may play a role in intracellular GABA(A) receptor transport but not synaptic anchoring, via its ability to interact with NSF. GABARAP may therefore have an important role in the production of GABAergic synapses.