14—3—3蛋白与肿瘤

一、14—3—3蛋白慨述 14-3-3蛋白是一种酸性可溶性蛋白,于1967年由Moor和Perez从牛的脑组织中分离,并因其经二乙氨乙基纤维素层析中的组分分布数量和在淀粉-凝胶电泳中的迁移位置而得名。由于14—3—3蛋白在脑组织中的含量最高,约占脑可溶性蛋白的1％,因而在很长一段时间内,它被认为是脑特异性蛋白,并且是动物所特有的、对维持脑神经功能具有独一无二作用的蛋白质。     

14-3-3蛋白在人脑胶质瘤中的表达及生物学意义

目的检测14-3-3蛋白在人脑胶质瘤中的表达情况,探讨其在胶质瘤发生发展中的生物学意义。方法采用免疫组化亲和素-生物素过氧化物酶复合物(ABC)法检测5个胶质瘤细胞系(U251MG,U87MG,BT325,SHG44和C6)、121例人脑胶质瘤石蜡标本和10例正常脑组织中14-3-3蛋白的表达情况。分析14-3-3蛋白的表达在胶质瘤发生发展中的作用。结果在正常脑组织标本中,13-3-3蛋白主要表达于神经元的胞体和突起,仅在少数的胶质细胞中可见14-3-3蛋白的弱表达。然而,5个胶质瘤细胞系和绝大部分星形细胞瘤中可见14-3-3蛋白的阳性表达,其表达阳性率为:Ⅰ级78.6%(11/14),II级75%(18/24),III级76.2%(16/21),IV级80%(20/25)。不同恶性级别的星形细胞瘤中,14-3-3蛋白的阳性表达率无显著差别,但14-3-3蛋白的表达强度和范围有随肿瘤的恶性度增高而增加的趋势。其它类型的胶质瘤中也可见14-3-3蛋白的大量表达,其表达阳性率为:少突胶质细胞瘤66.7%(4/6),间变型少突胶质细胞瘤100%(4/4),室管膜瘤50%(2/4),间变型室管膜瘤66.7%(2/3),脉络从乳头状瘤100%(5/5),松果体细胞瘤100%(3/3),髓母细胞瘤66.7%(8/12)。结论14-3-3蛋白在人脑胶质瘤中有大量的表达。14-3-3蛋白表达上调可能是胶质瘤细胞对抗调亡的一种共同机制,以14-3-3蛋白为靶点有望成为一种有前景的新的胶质瘤生物学治疗方法。     

脑脊液14—3—3蛋白检测在中枢神经系统疾病中的诊断价值

目的：本研究旨在通过对临床可能（可疑）克雅病（CJD）病例及其他神经系统病变患者的150份脑脊液标本进行14-3-3蛋白检测，对其临床意义和临床应用价值进行初步探讨。方法：用Westernblot方法检测脑脊液标本中的14—3—3蛋白。结果：150份标本中共19例阳性，其中临床诊断CJD者（n=6），5例阳性；临床疑似CJD者（n=23），6例阳性；非CJD痴呆者（n=6），1例阳性；单纯疱疹病毒性脑炎（n=7），3例阳性；其他病毒性脑膜脑炎、结核性脑膜脑炎、新型隐球菌脑膜炎、脑梗死各有1例阳性。结论：在临床诊断及疑似CJD病例中，脑脊液14—3—3蛋白检测是重要的实验室诊断手段；脑脊液14—3-3蛋白在CJD的诊断中强调病例的选择性。     

吉兰-巴雷综合征脑脊液14—3—3蛋白与脑脊液蛋白关系的研究

目的观察吉兰-巴雷综合征（GBS）脑脊液（CSF）中14—3—3蛋白与脑脊液蛋白在病程中的变化情况,探讨两者的相关性。方法采用酶联免疫吸附试验法（ELISA）检测43例GBS患者发病后3d内、第1,2,4,12周的脑脊液14—3—3蛋白含量,并同时用罗氏P-800全自动生化分析仪检测脑脊液蛋白含量。观察脑脊液14—3—3蛋白与脑脊液蛋白是否具有相关性。结果GBS组在发病3d内、第1,2,4,12周脑脊液14—3—3蛋白含量分别为（6．339±2．356）、（8．569±3．454）、（14．561±6．621）、（9．563±3．486）、（6．262±2．643）ng／ml,脑脊液蛋白分剐为（O．336±0．126）、（O．368±0．102）、（0．653±0．246）、（0．446±0．178）、（0．364±0．158）g／L。GBS组脑脊液14—3—3蛋白第1、2、4周高于对照组,差异有统计学意义（P〈0．05）,GBS组脑脊液蛋白第2、4周高于对照组,差异有统计学意义（P〈0．05）,脑脊液14—3—3蛋白含量与脑脊液蛋白含量存在正相关关系（P〈0．05）。结论GBS脑脊液中14—3—3蛋白含量的变化可引起脑脊液蛋白含量发生相应变化。     

14-3-3蛋白在星形细胞瘤中的表达及意义

目的探讨14-3-3蛋白在星形细胞瘤中的表达与肿瘤病理分级和预后间的关系。方法采用免疫组化ABC法检测10例正常脑组织和67例确诊并有随访的人脑星形细胞瘤石蜡标本中14-3-3蛋白的表达情况。分析14-3-3蛋白的表达与肿瘤恶性程度及预后间的关系。结果在正常脑组织标本中，14-3-3蛋白主要表达于神经元胞体和突起，而在少数的胶质细胞中仅见其弱表达。绝大部分星形胶质细胞瘤中可见14-3-3蛋白阳性表达，其阳性表达率为：Ⅱ级76．5％（13／17），Ⅲ级76．2％（16／21），Ⅳ级79．3％（23／29）。不同恶性级别的星形细胞肿瘤中，14-3-3蛋白的阳性表达率无显著差别（P〉0．05），但14-3-3蛋白表达的强度和范围有随肿瘤的恶性度增高而增加的趋势（P〈0．05）。52例14-3-3蛋白阳性表达患者的生存期明显短于15例14-3-3蛋白表达阴性的患者（P〈0．01）。结论14-3-3蛋白在人脑星形细胞瘤中的表达上凋与肿瘤的恶性程度及预后相关。14-3-3蛋白有望成为星形细胞瘤基因治疗的新靶点。     

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.     

14-3-3和CLIC4蛋白在U251细胞自噬中的相互作用

目的通过饥饿诱导神经胶质瘤U251细胞发生自噬,探讨细胞CLIC4和14-3-3蛋白在饥饿条件下诱导自噬过程中的相互作用。方法通过Hoechst、14-3-3 epsilon、CLIC4染色于共聚焦显微镜下观察抑制CLIC4表达对于饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位的影响。通过Western Blot技术检测Beclin 1及14-3-3蛋白表达。免疫共沉淀技术检测14-3-3 epsilon蛋白与CLIC4蛋白的结合水平。结果共聚焦显微镜观察14-3-3 epsilon和CLIC4荧光染色结果显示,饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位显著增加,并广泛分布于胞浆及细胞核中。同时Western Blot结果表明抑制CLIC4表达能够引起14-3-3蛋白以及自噬相关蛋白Beclin1表达增加。饥饿条件下,14-3-3 epsilon蛋白与CLIC4共沉淀增强,而抑制CLIC4表达能够降低两者结合水平。结论 14-3-3epsilon蛋白与CLIC4的相互作用由于RNA干扰而减弱,促进了14-3-3蛋白水平上调,进而增强了14-3-3蛋白对Beclin1信号通路的调节,引起Beclin1表达增加,进一步激活饥饿条件下U251细胞自噬过程。     

14-3-3蛋白在神经疾病中的临床研究

114-3-3蛋白的生理特性和检测方法14-3-3蛋白是高度保守的酸性蛋白家族,其分子量在25～30Kda,有7种亚型,分别为β、ε、γ、η、σ及τ,由不同的基因编码,各亚型之间有高度的同源性。它们几乎在所有的组织中表达,在神经系统中含量最丰富,约占全部可溶性蛋白质的1%。所有神经元、星形胶质细胞、少突胶质细胞、小胶质细胞的胞浆与胞核中都有表达。14-3-3蛋白对众多的靶蛋白具有调节作用,主要依赖于磷酸化方式。14-3-3蛋白主要参与信号转导、细胞内蛋白质的转运、细胞增殖周期和凋亡的调节、细胞骨架构建等,尚有一些作用目前还不清楚。1.114-3…     

14-3-3蛋白过表达减轻1-甲基-4-苯基吡啶离子对PC12细胞的毒性

目的 研究14—3—3蛋白过表达对1-甲基-4苯基吡啶离子（MPP^＋诱导的PC12细胞死亡的影响作用及其可能的机制。方法 构建pcDNA3．1（＋）-14—3—3真核表达质粒,用脂质体2000转染PCI2细胞;Westernn blot技术检测PC12细胞中14—3—3蛋白、Bcl-2蛋白,和BAD蛋白的表达;然后分别用MTT法、酶标仪及流式细胞仪检测PC12细胞的活力、caspase的活性及PC12细胞的凋亡率。结果 （1）将pcDNA3．1（＋）-14—3—3质粒转染PCI2细胞3周后,14—3—3蛋白的表达显著增加;（2）MPP^＋诱导PC12细胞存活率的下降是剂量依赖性的,当MPP^＋的浓度达100μmol／L时,PC12细胞的存活率丧失约50％;（3）caspase的活性随着MPP^＋浓度的增加而增高,当MPP^＋浓度到达100μmol／L时caspase的活性也到达最大值,而当MPP^＋浓度超过100μmol／L时,caspase的活性急剧下降;（4）用100μmol／L的MPP^＋处理PC12细胞24h后,PC12细胞的凋亡率为26．5％,14—3—3蛋白的过表达使PC12细胞的凋亡率下降到8．6％;（5）用100μmol／LMPP^＋处理PC12细胞后,Bcl-2蛋白的表达趋于下调而BAD蛋白的表达上调,14—3-3蛋白的过表达能显著的增加Bcl-2蛋白的表达而使BAD蛋白的表达下调。结论 14—3—3蛋白过表达通过上调Bcl-2蛋白的表达并下调BAD蛋白的表达,减少了MPP^＋诱导的PC12细胞的凋亡,从而发挥对PC12细胞的保护作用。这些结果可能为PD的治疗提供新的药物靶点。     

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。     

Isoform-specific expression of 14-3-3 proteins in human astrocytoma

BACKGROUND: 14-3-3 protein plays crucial roles in tumorigenesis, including the maintenance of cell cycle and DNA repair, the prevention of apoptosis, among others. In mammalian cells, seven 14-3-3 isoforms (beta, epsilon, zeta, eta, theta, gamma and sigma) have been identified and each of these seems to have distinct tissue localizations and isoform-specific functions. In the present study, the levels of all seven 14-3-3 isoforms were examined in astrocytoma. METHODS: The expression of 14-3-3 isoforms and their protein expression levels were examined in five glioma cell lines by western blotting. Then in astrocytoma tissues, we investigated expression percentages of each isoform by immunohistochemistry. The protein and mRNA expression levels of each isoform were also detected by western blotting and RT-PCR, respectively. RESULTS: 14-3-3beta and eta were specifically expressed in astrocytoma, and their expression frequencies and levels increased with the increase of astrocytoma malignancy. The result from glioma cell lines was consistent with that from astrocytoma tissue. CONCLUSIONS: In our study, we found two tumor-specific isoforms of 14-3-3 in astrocytoma. They might be involved in astrocytoma tumorigenesis and may be useful as targets for therapy.     

Distribution of S-100 and 14-3-2 proteins on neuronal cell membranes.

The distribution of nervous tissue specific S-100 and 14.3.2 proteins was studied over the total surface of isolated, intact nerve cells by immunofluorescence microscopy. The distribution of the antigens was different on the front and back of the cells, indicating complicated S-100 and 14.3.2 patterns. A partly overlapping in membrane pattern of S-100 and 14.3.2 proteins was observed, although S-100 protein dominated with respect to membrane areas covered. At higher resolution the specific fluorescence appeared as conglomerates and islets. The neuronal membrane patterns of S-100 and 14.3.2 antigens, including synapses suggest that nerve cell membranes are functionally differentiated, thus greatly increasing the capacity for identification of incoming stimuli.     

Enhanced expression of 14-3-3 proteins in reactive astrocytes in Creutzfeldt-Jakob disease brains

14-3-3 proteins have been reported to be detected specifically in the cerebrospinal fluid (CSF) from patients with Creutzfeldt-Jakob disease (CJD). To elucidate the role of 14-3-3 proteins in patients with CJD, we performed immunohistochemical studies on 14-3-3 proteins in autopsied brains from five patients with sporadic CJD (sCJD), three patients with Alzheimers disease (AD), and seven normal control subjects. Formalin-fixed, paraffin-embedded sections from all cases were immunostained with several types of specific anti-14-3-3 antibodies. In the normal control brains, 14-3-3 immunoreactivity was localized mainly in the neuronal somata and processes; in contrast, glial cells showed no or faint immunoreactivity. In the brains from the patients with AD, 14-3-3 immunoreactivity was observed in the surviving neurons as well as some neurofibrillary tangles. In the brains from the patients with sCJD, 14-3-3 immunoreactivity was well preserved in the remaining neurons. Furthermore, the glial cells, especially the reactive astrocytes, were intensely immunostained in the brains affected by sCJD. Our findings suggest that 14-3-3 proteins may be up-regulated in the glial cells, particularly in reactive astrocytes, and that the enhanced expression of 14-3-3 proteins in these glial elements may be associated with the pathogenesis of sCJD.     

Accumulation of 14-3-3 proteins in glial cytoplasmic inclusions in multiple system atrophy

Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy. However, the molecular mechanisms underlying the formation of glial cytoplasmic inclusions remain unclear. Alpha-synuclein, a major component of glial cytoplasmic inclusions, has the ability to interact with 14-3-3 proteins, which mediate several types of signal transduction pathways. To elucidate the role of these 14-3-3 proteins in patients with multiple system atrophy, we performed immunohistochemical studies on 14-3-3 in brain tissue specimens from 7 control subjects and from 15 patients with multiple system atrophy. In both control and multiple system atrophy cases, 14-3-3 immunoreactivity was observed mainly in the neuronal somata and proximal processes, as well as the nerve fibers. Even in the severely affected regions of patients with multiple system atrophy, 14-3-3 immunoreactivity generally was spared in the surviving neurons, some of which were strongly immunolabeled. In addition, numerous glial cytoplasmic inclusions were intensely immunostained, and neuronal cytoplasmic inclusions and dystrophic neurites were also immunoreactive for 14-3-3. Our results suggest that an aberrant accumulation of 14-3-3 proteins may occur in brains affected by multiple system atrophy, and that 14-3-3 proteins may be associated with the pathogenesis of multiple system atrophy.     

Quantitation of 14-3-3 and neuron-specific enolase proteins in CSF in Creutzfeldt-Jakob disease

CSF 14-3-3 and neuron-specific enolase (NSE) proteins were quantitated from patients who had Creutzfeldt-Jakob disease (CJD) or other rapidly dementing disorders initially considered to be CJD. Thirty-one patients were diagnosed as having CJD among 152 studied. CSF 14-3-3 values more than 8 ng/mL correlated with CJD. CSF NSE values less than 30 ng/mL and 14-3-3 values less than 8 ng/mL made a diagnosis of CJD unlikely, but did not exclude it.     

Rapid identification of 14-3-3-binding proteins by protein microarray analysis

The 14-3-3 protein family consists of acidic 30-kDa proteins composed of seven isoforms in mammalian cells, expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 isoforms form a dimer that acts as a molecular adaptor interacting with key signaling components involved in cell proliferation, transformation, and apoptosis. Until present, more than 300 proteins have been identified as 14-3-3-binding partners, although most of previous studies focused on a limited range of 14-3-3-interacting proteins. Here, we studied a comprehensive profile of 14-3-3-binding proteins by analyzing a high-density protein microarray using recombinant human 14-3-3 epsilon protein as a probe. Among 1752 proteins immobilized on the microarray, 20 were identified as 14-3-3 interactors, most of which were previously unreported 14-3-3-binding partners. However, 11 known 14-3-3-binding proteins, including keratin 18 (KRT18) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), were not identified as a 14-3-3-binding protein. The specific binding to 14-3-3 of EAP30 subunit of ELL complex (EAP30), dead box polypeptide 54 (DDX54), and src homology three (SH3) and cysteine rich domain (STAC) was verified by immunoprecipitation analysis of the recombinant proteins expressed in HEK293 cells. These results suggest that protein microarray is a powerful tool for rapid and comprehensive profiling of 14-3-3-binding proteins.     

14-3-3蛋白与阿尔茨海默病tau蛋白异常磷酸化的关系

目的研究阿尔茨海默病(Alzheimerdisease,AD)患者海马组织1433蛋白家族与神经原纤维缠结(NFT)形成及tau蛋白异常磷酸化之间的关系。方法以4例临床及病理确诊的AD患者及4名对照者海马组织为材料,用免疫组织化学、免疫印迹及免疫共沉淀技术来研究1433蛋白在AD患者海马的表达情况及其与异常磷酸化tau蛋白之间的关系。结果1433蛋白ζ及γ亚型能使AD患者海马的NFT显色,β亚型主要使海马的颗粒神经元着色,同时也使少许NFT染色。免疫印迹显示β和ζ亚型在AD中明显上调,其像素值(分别为4570±92和5630±40)比对照组(分别为1849±65和956±140)高2～3倍(P<0.01,t检验)。免疫共沉淀分析发现单抗双螺旋细丝(PHF1)从AD患者脑中沉淀的1433蛋白像素值(3859±78)显著多于对照组(1540±138,AD组像素值比对照组高1倍,P<0.05,t检验),这3种亚型均与异常磷酸化tau蛋白在AD患者脑部形成复合物。σ亚型在AD患者和对照组脑中均无表达。上述发现在4名对照者脑组织中并未观察到。结论β、ζ及γ亚型1433蛋白参与NFT形成,它们均可能参与tau蛋白的磷酸化调节,具体机制有待阐明。     

14-3-3 proteins and protein phosphatases are not reduced in tau-deficient mice

Tau is an axonal microtubule-associated protein, whose dysfunction causes neurodegenerative diseases such as Alzheimer's disease and other tauopathies. Earlier studies have shown the interactions of tau with glycogen synthase kinase-3beta, 14-3-3zeta, protein phosphatase 1 and protein phosphatase 2A. In this study, we compared the amounts of these tau-interacting proteins in brain microtubule-enriched fractions from wild-type and tau-deficient mice. Contrary to our expectation, we detected no difference in the amount of these proteins between wild-type and tau-deficient mice. Our findings indicate that only a small portion of tau-interacting proteins are bound to tau in vivo, and suggest the existence of other scaffolding proteins. We propose that tau-deficient mice are an ideal system for confirming the function of tau-interacting proteins.