糖元合酶激酶-3过度激活对神经细丝磷酸化的影响

目的：在细胞水平研究糖元合酶激酶((3SK-3)过度激活对神经细丝磷酸化的影响。方法：采用磷酯酰肌醇三磷酸激酶(P13K)的特异性抑制剂渥曼青霉素(wortmannin,WT)处理野生型小鼠成神经瘤细胞株(N2a／wt),免疫印迹技术及酶活性测定检测GSK-3活性,免疫荧光技术检测神经细丝(NF)的磷酸化状态。进一步采用(3SK-3特异性的抑制剂LiCl处理上述细胞,检测上述相同指标。结果：WT处理N2a细胞1h后,GSK3酶活性显著增加,并使抗体SMI31显色增强,SMI32显色减弱,提示NF被过度磷酸化。而采用WT和LiCl联合处理N2a／wt细胞,GSK-3活性显著降低,与此同时,NF的磷酸化程度明显减少。结论：GSK-3过度激活可引起细胞骨架蛋白NF的异常过度磷酸化,而过度磷酸化的NF可能参与了AD的病理过程。     

miR-124-3p通过负调控Caveolin-1表达降低N2a/APPswe细胞内Tau蛋白磷酸化水平

目的探讨在阿尔茨海默病(AD)细胞模型中miR-124-3p通过调控Caveolin-1的表达对细胞内Tau蛋白磷酸化水平的影响。方法体外培养野生型N2a(N2a/WT)和N2a/APPswe细胞,荧光定量PCR及Western blot分别检测miR-124-3p、APP和Caveolin-1的表达;N2a/APPswe细胞分别转染miR-124-3p模拟物、Caveolin-1过表达载体及干扰RNA后,用荧光定量PCR及Western blot分别检测Caveolin-1、Tau和Tau-Ser404表达。结果与野生型N2a细胞比较,N2a/APPswe细胞中miR-124-3p表达降低(P0.01),APP表达升高(P0.01),Caveolin-1表达升高(P0.01)。N2a/APPswe细胞转染miR-124-3p模拟物后,Caveolin-1表达降低(P0.01),Tau-Ser404/Tau降低(P0.01)。N2a/APPswe细胞转染Caveolin-1过表达载体后,Tau-Ser404/Tau升高(P0.01)。转染干扰RNA后TauSer404/Tau降低(P0.01)。结论 miR-124-3p可能通过调节其靶基因Caveolin-1的表达而降低Tau蛋白磷酸化水平,在AD中发挥神经保护作用。     

稳定表达GHR基因和突变体的CHO细胞株的构建及其功能分析

目的构建稳定表达先天性生长激素不敏感综合征相关基因-hGHR及突变体(hGHR-E42K、hGHR-H56R)的CHO细胞株,测定野生株和突变株的STAT5-磷酸化水平。方法利用已有的PUC-hGHR质粒,定点突变获得2个hGHR突变体(hGHR-E42K、hGHR-H56R),限制性酶切法将目的基因克隆到真核表达载体pcDNA3.1/(zeo+);然后用Lipofectamine2000将重组子转染至CHO细胞,通过Zeocin抗性筛选稳定表达hGHR及突变体的细胞群;并用RT-PCR和Western blot证实hGHR、STAT5-P表达水平。结果测序验证hGHR的E42K和H56R错义突变成功引入,并克隆到真核表达载体;转染后在CHO细胞株中成功检测到hGHR和突变体的mRNA和蛋白;突变细胞株(E42K、H56R)的磷酸化STAT5蛋白表达量低于野生株。结论成功构建携带稳定表达hGHR及突变体的CHO细胞株,E42K、H56R突变部分阻碍STAT5蛋白进行磷酸化。     

ARK5与乳腺癌侵袭转移关系的研究

目的探讨ARK5在乳腺癌侵袭和转移中的意义。方法采用免疫组化SP法检测58例乳腺癌组织和15例癌旁乳腺组织中MMP-2、MMP-9及ARK5表达。应用小RNA干扰技术,将合成的小RNA干扰质粒转染MDA-MB-435细胞株,采用Western blot法检测瞬时转染后ARK5蛋白表达情况。通过体外侵袭实验检测细胞转染后侵袭能力的变化。ARK5下降后,再次进行Western blot法检测MMP-2、MMP-9蛋白表达。结果乳腺癌组织中MMP-2、MMP-9和ARK5免疫组化阳性结果之间存在正相关性。转染后的细胞株命名:转染ARK5质粒的MDA-MB-435细胞称之为SiARK5/MDA-MB-435,转染对照质粒的MDA-MB-435细胞称之为Scr/MDA-MB-435。转染72 h后,与Scr/MDA-MB-435细胞相比,SiARK5/MDA-MB-435细胞的ARK5蛋白表达水平降低。ARK5表达降低的乳腺癌细胞侵袭并穿透Matrivgel膜基质的细胞数量比对照组少(P<0.01),且MMP-2、MMP-9蛋白表达量降低。结论应用小RNA干扰技术降低ARK5蛋白的表达使MDA-MB-435细胞侵袭转移能力降低,同时MMP-2、MMP-9蛋白表达降低,提示ARK5通过MMP-2、MMP-9在乳腺癌侵袭转移中发挥重要作用。     

ARK5 is associated with the invasive and metastatic of breast carcinoma

目的 探讨ARK5在乳腺癌侵袭和转移中的意义.方法 采用免疫组化SP法检测58例乳腺癌组织和15例癌旁乳腺组织中MMP-2、MMP-9及ARK5表达.应用小RNA干扰技术,将合成的小RNA干扰质粒转染MDA-MB-435细胞株,采用Western blot法检测瞬时转染后ARK5蛋白表达情况.通过体外侵袭实验检测细胞转染后侵袭能力的变化.ARK5下降后,再次进行Western blot法检测MMP-2、MMP-9蛋白表达.结果 乳腺癌组织中MMP-2、MMP-9和ARK5免疫组化阳性结果之间存在正相关性.转染后的细胞株命名:转染ARK5质粒的MDA-MB-435细胞称之为SiARK5/MDA-MB-435,转染对照质粒的MDA-MB-435细胞称之为Scr/MDA-MB-435.转染72 h后,与Scr/MDA-MB-435细胞相比,SiARK5/MDA-MB-435细胞的ARK5蛋白表达水平降低.ARK5表达降低的乳腺癌细胞侵袭并穿透Matrivgel膜基质的细胞数量比对照组少(P<0.01),且MMP-2、MMP-9蛋白表达量降低.结论 应用小RNA干扰技术降低ARK5蛋白的表达使MDA-MB-435细胞侵袭转移能力降低,同时MMP-2、MMP-9蛋白表达降低,提示ARK5通过MMP-2、MMP-9在乳腺癌侵袭转移中发挥重要作用.     

Brn-3a诱导胚胎干细胞向神经样细胞定向分化的研究

目的　探讨转录因子Brn 3a诱导胚胎干细胞 (ES细胞 )向神经细胞定向分化的可能性。　方法　将带有目的基因 (Brn 3a转录因子 )的重组表达载体pJ5Brn 3a ,通过脂质体转染到小鼠ES细胞株中进行表达 ,促使ES细胞向神经细胞分化 ,并采用免疫组织化学技术检测转染前后转录因子Brn 3a蛋白及分化后具有神经元表型特征的神经样细胞特异抗原的表达。　结果　 1 转染前ES细胞Brn 3a免疫反应阴性 ,转染后 2 4h检测Brn 3a免疫反应呈阳性 ;2 转染细胞经 1周培养 ,70 %以上的细胞具有明显的神经细胞样突起 ,神经细胞特有标记抗原NFH L、NSE及SY呈免疫反应阳性 ;神经胶质细胞特有标记抗原GFAP为阴性。　结论　转录因子Brn 3a能成功诱导ES细胞向神经细胞定向分化     

miR-708-5p通过靶向URGCP抑制非小细胞肺癌A549细胞侵袭、转移的研究

目的 探讨miR-708-5p对非小细胞肺癌A549细胞的增殖、迁移和侵袭的影响及其与上调基因-4(upregulated gene-4/upregulator of cell proliferation,URG4/URGCP)的靶向关系.方法 选取非小细胞肺癌细胞株A549,正常肺成纤维细胞HLF-1为研究对象,根据A549细胞转染物质的不同,分为Control组(未进行任何处理),NC组(转染随机序列RNA Oligo),miR-708-5p组(转染miR-708-5p mimics).MTT检测增殖能力,流式细胞术检测细胞周期,Transwell小室实验检测侵袭能力,划痕实验检测迁移能力,Western blot检测URGCP表达水平;实时荧光定量PCR检测miR-708-5p、URGCP基因mRNA表达水平;荧光素酶实验验证miR-708-5p与URGCP的靶向关系.结果 与HLF-1细胞株相比,肺癌细胞株A549细胞miR-708-5p表达水平降低,URGCP-mRNA表达升高(P<0.05),同时,URGCP蛋白水平升高.转染72h后,与Control、NC组比较,miR-708-5p组细胞URGCP基因mRNA明显降低,URGCP蛋白表达量明显降低(P均<0.01).miR-708-5p组细胞增殖、迁移和侵袭能力明显低于Control、NC组(P<0.05).与WT-URGCP组相比,miR-708-5p+WT-URGCP组细胞荧光素酶活性明显降低(P<0.01).结论 miR-708-5p在非小细胞肺癌A549细胞中低表达,过表达miR-708-5p可抑制A549细胞增殖、迁移和侵袭,机制可能与负性调控URGCP有关.     

Raptor对胶质瘤细胞侵袭能力的影响

 目的：研究Raptor对于胶质瘤细胞侵袭能力的影响。方法：采用RNA干扰技术,向胶质瘤U87细胞转染Raptor限定性siRNA干扰质粒, Western blotting检测转染后细胞Raptor的表达水平以鉴定转染效果。体外侵袭实验检测Raptor表达降低后,U87细胞侵袭能力的变化;Western blotting检测细胞中ARK5的磷酸化情况和MMP-2、MMP-9的表达水平。免疫组织化学法检测低级别及高级别胶质瘤中Raptor的表达水平。结果：转染Raptor siRNA质粒的U87细胞命名为siRaptor/U87,转染对照组质粒的细胞命名为Scr/U87,转染成功的实验组细胞Raptor的表达降低。体外侵袭实验中siRaptor/U87较对照组穿透基质膜的细胞数少(P＜0.01)。Western blotting显示实验组细胞中磷酸化ARK5、MMP-2和MMP-9蛋白的表达水平均较对照组低。胶质瘤组织中Raptor的表达与恶化程度存在相关性（P＜0.01）。结论：Raptor 表达降低可能通过磷酸化ARK5及增加MMP-2、MMP-9的表达促进胶质瘤细胞的侵袭力。     

miR-155-5p靶向下调SOCS1减轻脂多糖诱导的SH-SY5Y人神经母细胞瘤细胞炎症损伤

目的 探讨微小RNA-155-5p(miR-155-5p)对脂多糖(LPS)诱导SH-SY5Y人神经母细胞瘤细胞神经炎症损伤的影响。方法 采用人SH-SY5Y细胞，设立miR-155-5p过表达及其阴性对照(miR-155-5p mimic组、mimic-NC组)和miR-155-5p低表达及其阴性对照(miR-155-5p inhibitor组、inhibitor-NC组),将LPS处理上述各组转染成功细胞24 h,并设置未转染SH-SY5Y细胞的对照组和LPS处理组(LPS组)。噻唑蓝(MTT)法检测SH-SY5Y细胞活性，流式细胞术检测细胞凋亡率，反转录PCR法检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6和IL-10 mRNA水平和miR-155-5p表达，Western blot法检测细胞裂解型胱天蛋白酶3(c-caspase-3)、B细胞淋巴瘤因子2(Bcl2)、Bcl2相关X蛋白(BAX)、磷酸化核因子κB p65/核因子κB p65(p-NF-κB p65/NF-κB p65)、磷酸化p38丝裂原活化蛋白激酶/p38丝裂原活化蛋白激酶(p-p3...     

miR-449a过表达抑制人神经母细胞瘤细胞SH-SY5Y增殖并促进其凋亡

目的探讨miR-449a对人神经母细胞瘤细胞系SH-SY5Y的增殖和凋亡的影响。方法用Lipofectamine TM2000将miR-449a模似物或miR-449a对照转染至SH-SY5Y细胞,分为空白、miR-449a模似物和miR-449a对照SHSY5Y细胞组;实时荧光定量PCR(q-PCR)检测各组细胞中miR-449a表达;CCK8法检测细胞增殖;流式细胞仪检测细胞凋亡和周期,Western blot检测c-Myc蛋白和Bax/Bcl-2蛋白表达。结果 miR-449a模似物瞬时转染SH-SY5Y细胞后,miR-449a的表达水平明显高于正常对照组(P0.05);SH-SY5Y细胞增殖能力受到明显抑制(P0.05);凋亡率明显增加(P0.05);c-Myc蛋白表达显著降低(P0.05);细胞促凋亡蛋白Bax表达升高;抗凋亡蛋白Bcl-2表达降低(P0.05)。结论 miR-449a可通过c-Myc影响SH-SY5Y细胞的增殖和周期,通过调节Bax/Bcl-2影响其凋亡。     

Expression and phosphorylation of neurofilament protein in different neuronal tissues

Expression and phosphorylation of neurofilament protein in different neuronal tissues     

Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer brain and the possible mechanism

Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer brain and the possible mechanism     

Calpain mediates calcium-induced activation of the erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons: relevance to Alzheimer's disease

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimer's disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.     

Declining phosphatases underlie aging-related hyperphosphorylation of neurofilaments

Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.     

Distribution and expression of developmentally regulated phosphorylation epitopes on MAP 1B and neurofilament proteins in the developing rat spinal cord

Summary The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstrationin vivo of a mAb 150 axonal gradient similar to that seen previouslyin vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies.     

急、慢性吗啡处理对大鼠大脑皮层tau蛋白和神经微丝磷酸化水平的影响

目的:观察急、慢性吗啡处理对大鼠大脑皮质细胞骨架蛋白磷酸化水平的影响,探讨吗啡导致细胞周期依赖性蛋白激酶-5(cyclin dependent kinase-5,CDK5)过度表达与细胞骨架蛋白过度磷酸化的关系。方法:雄性SD大鼠40只,随机均分为急性对照组、急性吗啡处理组、慢性对照组、慢性吗啡处理组。急性吗啡处理组腹腔注射吗啡30mg/kg1次,慢性吗啡处理组腹腔注射吗啡10mg/kg,每天2次(时间8:00、20:00)共10d。采用免疫印迹法测定大鼠大脑皮质tau蛋白和神经微丝的磷酸化水平及CDK5、p35表达水平。结果:(1)与急性对照组比较,急性吗啡处理组tau蛋白和神经微丝磷酸化水平升高,CDK5的表达增加;(2)与慢性对照组比较,慢性吗啡处理组tau蛋白和神经微丝磷酸化水平升高,CDK5的表达无明显变化;(3)与急性吗啡处理组比较,慢性吗啡处理组tau蛋白和神经微丝蛋白磷酸化水平降低;(4)各组间p35表达水平无明显改变。结论:急、慢性吗啡处理可导致大鼠大脑皮质tau蛋白和神经微丝的异常过度磷酸化,但这种变化可能与CDK5的过度表达无关。     

The primary auditory cortex in cetacean and human brain: a comparative analysis of neurofilament protein-containing pyramidal neurons.

To extend our investigation of the anatomy of sensory systems in highly adapted aquatic and terrestrial mammals, we have analyzed the distribution of a particular population of efferent neurons in the cetacean and human primary auditory cortex using an antibody to non-phosphorylated neurofilament protein (SMI32). The neurofilament protein triplet is differentially distributed within neuronal subpopulations in the primate and cetacean neocortex. In primates, it appears that the somatodendritic domain of a subset of pyramidal neurons furnishing specific corticocortical connections contains high concentrations of neurofilament protein. In the human primary auditory cortex these neurons are located in layers III, V and VI, whereas in cetaceans they are concentrated almost exclusively in the cortical efferent layer IIIc/V. Previous analyses have shown that SMI32 immunoreactivity in the cetacean neocortex is uniformly distributed among functionally different areas, while in human neocortex, the distribution of SMI32-positive neurons exhibit a high degree of regional and laminar specialization that is correlated with the functional and anatomical diversity of the cortical areas. In addition, the overall distribution of SMI32-immunoreactive neurons in the cetacean neocortex is comparable to that observed in paralimbic areas of the human, suggesting that the cetacean neocortex has retained many features of phylogenetically older cortical regions.     

An ATPase activity associated with the rotavirus phosphoprotein NSP5

Interactions between NSP5 and NSP2 drive the formation of viroplasms, sites of genome replication and packaging in rotavirus-infected cells. The serine-threonine-rich NSP5 transitions between hypo- and hyper-phosphorylated isomers during the replication cycle. In this study, we determined that purified recombinant NSP5 has a Mg2+-dependent ATP-specific triphosphatase activity that generates free ADP and Pi (Vmax of 19.33 fmol of product/min/pmol of enzyme). The ATPase activity was correlated with low levels of NSP5 phosphorylation, suggestive of a possible link between ATP hydrolysis and an NSP5 autokinase activity. Mutagenesis showed that the critical residue (Ser67) needed for NSP5 hyperphosphorylation by cellular casein kinase-like enzymes has no role in the ATPase or autokinase activities of NSP5. Through its NDP kinase activity, the NSP2 octamer may support NSP5 phosphorylation by creating a constant source of ATP molecules for the autokinase activity of NSP5 and for cellular kinases associated with NSP5.     

Identification and characterization of SMI32-immunoreactive amacrine cells in the mouse retina

Mammalian neurons express the neural intermediate filament protein neurofilament (NF). In the retina, NFs have been detected primarily in the axons and processes of retinal ganglion and horizontal cells. We found an amacrine cell type that was immunolabeled with an antibody against SMI32, a non-phosphorylated epitope on neurofilament proteins of high molecular weight, in the mouse retina. This type of amacrine cell was non-randomly distributed, and these cells exhibited a central-peripheral density gradient. Most of these cells co-expressed GABA and ChAT, but not glycine or any other amacrine cell marker. These results suggest that some SMI32-immunoreactive amacrine cells belong to a GABAergic population, and that SMI32 can therefore be used as a marker for a subset of amacrine cells in addition to ganglion cells and horizontal cells in the mouse retina.