连环蛋白家族成员P120ctn的作用及相关机制

连环蛋白P120可在细胞连接处与E-钙黏蛋白结合形成连环蛋白-钙黏蛋白复合体,调控钙黏蛋白介导的细胞黏附作用;在胞质内可与Rho家族GTP酶相互作用调节细胞骨架的运动;在细胞核内可与核转录因子NF-κB和转录抑制因子Kaiso结合,影响炎性反应和细胞增殖.P120对细胞黏附、细胞动力、炎性反应和细胞增殖的影响使其与损伤...     

连环蛋白家族新成员——P120ctn

连环蛋白家族最早被分为α、β、γ3类,与E-钙粘蛋白共同作用参与细胞黏附。P120连环蛋白作为新发现的Cat家族成员之一,在保持细胞黏附中起重要作用,参与了胚胎组织和神经系统形成,同时也参与肿瘤发生发展,其表达缺失或下降将导致细胞分化丧失和具有侵袭性,容易发生浸润和转移。     

P120ctn与肿瘤

P120ctn是一种新发现的位于细胞间连接处和细胞核内的连环素(catenin).具有与α,β,γ连环素类似的结构,但其功能存在差异.P120ctn通过与高度保守的钙粘附素(cadherin)的近膜区域(juxtamembrane domain,JMD)相互作用而影响细胞的粘附.在核内可能与信号转导有关.P120ctn在恶性肿瘤中有异常表达,提示其在恶性肿瘤的浸润、转移中具有一定的作用.     

E-cadherin表达缺失在白血病细胞行为中的作用

目的:确定上皮-钙黏蛋白(E-cadherin)表达缺失在白血病细胞行为中的作用,最终阐明E-cadherin介导的骨髓微环境与造血细胞相互作用改变在白血病发生中的作用。 方法:细胞黏附实验测定 HL-60细胞经5-氮杂脱氧胞苷（5-Aza-CdR）诱导E-cadherin表达后细胞黏附能力的改变,MTT法测定细胞增殖能力,细胞迁移实验确定细胞的迁移能力。结果:HL-60细胞经5-Aza-CdR诱导E-cadherin表达后,其对E-cadherin-Fc的黏附能力增加,细胞在E-cadherin-Fc上的增殖能力下降,细胞黏附导致细胞迁移能力下降。结论:白血病细胞中由于E-cadherin表达丧失,引起细胞黏附降低,增殖能力增强,细胞迁移活跃,表明E-cadherin表达缺失与白血病细胞行为的获得有关。     

再生丝素蛋白膜表面的纳米图案化修饰对细胞黏附和增殖的影响

为了拓展再生丝素蛋白(RSF)生物医用材料在细胞培养和组织修复等生物医学领域中的应用,提高RSF材料的细胞相容性至关重要。本研究采用等离子体刻蚀技术制备了纳米“岛”图案化RSF膜,并进一步考察其对NIH3T3细胞黏附和增殖行为的影响。所有定量检测实验中至少设3个重复样(n≥3)。细胞培养1 d后的显微观察结果表明,与未修饰的RSF平整膜相比,纳米图案化修饰的RSF膜具有更好的黏附形态。CCK-8检测细胞培养1和4 d的吸光度(OD)结果表明,与未修饰的RSF平整膜相比,纳米图案化修饰RSF膜能够明显促进细胞的增殖,且其中刻蚀30 min膜组的吸光度(1.13±0.32)最高,显著高于未修饰的RSF平整膜(0.46±0.03,P<0.001)。等离子体刻蚀技术可作为一种简便、快捷、可大规模制备的纳米图案化修饰策略用于提高RSF材料的细胞相容性。     

细胞连素和钙调节素在发育生物学中的作用

细胞连素和钙调节素在胚胎形态发生中是关键的分予，最近的研究表明，细胞连素和钙调节素涉及到信号传递的通路。本文从三个方面阐述了细胞连素和钙调节素在胚胎形态发生中的调节功能。     

查菲埃立克体表面抗原P120蛋白基因的克隆与表达

查菲埃立克体 (Ｅｈｒｌｉｃｈｉａｃｈａｆｆｅｅｎｓｉｓ)是人单核细胞埃立克体病 (ｈｕｍａｎｍｏｎｏｎ ｃｙｔｉｃｅｈｒｌｉｃｈｉｏｓｉｓ,ＨＭＥ)的病原体 ,已证实我国存在该病原。血清学分析是目前诊断ＨＭＥ及其流行病学调查的主要方法。但由于该病原体为严格胞内寄生菌 ,难以通过细胞培养制备大量抗原去满足临床ＨＭＥ的血清学诊断和流行病学调查的需要。Ｐ12 0 (相对分子质量为 12 0×10 3蛋白 )为查菲埃立克体主要表面抗原 ,具有种特异性。该蛋白基因包含了多个连续的 2 40ｂｐ重复单位 ,占全基因的 6 0 % ,并均为Ｂ淋巴细胞识…     

P53在细胞缺氧调控中的作用

缺氧损伤是常见而又重要的病理过程,P53蛋白在缺氧调控中发挥的作用也逐渐为人们所认识。P53可在细胞核内富集并协助细胞快速应答缺氧信号,还能根据缺氧程度等因素间接决定缺氧损伤细胞的命运,促使细胞周期停滞、衰老或凋亡等。因此,P53是细胞缺氧损伤与保护过程中的重要影响因素。     

Rac1介导E-cadherin下调引起的白血病细胞行为改变

目的:确定上皮-钙黏蛋白(E-cadherin)表达下调或缺失在白血病细胞恶性行为中的作用及分子机制,最终阐明骨髓微环境与造血细胞相互作用改变在白血病发生中的作用。方法:通过RNA干扰白血病细胞(Raji细胞)及基质细胞(293T细胞)中E-cadherin的表达;细胞黏附实验测定E-cadherin表达沉默后2种细胞间的黏附能力改变;MTT法测定干扰后293T细胞上Raji细胞的增殖能力;Westernblotting方法确定干扰后Rho家族小GTP酶Rac1的表达。结果:RNA干扰E-cadherin表达后,Raji细胞对293T细胞的黏附能力显著降低,在293T细胞上的增殖能力显著增强,同时Rac1的表达升高。结论:白血病细胞中E-cadherin表达丧失,引起细胞黏附降低,增殖能力增强,表明E-cadherin表达缺失与白血病细胞恶性行为的获得有关,Rac1可能介导了E-cadherin表达缺失引起的细胞行为改变。     

P53基因对K562细胞增殖的影响

目的 观察恢复K562细胞的p53蛋白功能后该细胞的生物学行为的改变，探索用野生型p53基因治疗白血病的可能性。方法 以K562细胞为研究对象，电穿孔法转导野生型p53基因，RT-PCR和免疫细胞化学方法检测p53基因的表达，^3H-TdR掺入法检测细胞增殖，用流式细胞仪分别以TUNEL、annexin-V、细胞周期检测细胞的凋亡情况和细胞周期分布情况。结果 转染p53基因的K562细胞，出现明显细胞周期G1期停滞，增殖抑制率为24．17％，但用TUNEL、annexin-V和亚二倍体峰检测未发现与未转染p53基因组有凋亡细胞比例差异。结论 P53基因对K562细胞有一定的治疗效应，但不能诱导其发生凋亡。     

生物信息学在蛋白质结构与功能预测中的应用

生物信息学是现代生命科学与信息科学、计算机科学、数学、统计学、物理学、化学等学科相互渗透而高度交叉形成的一门新兴前沿学科。随着人类基因组计划的完成,应用生物信息学技术预测蛋白质结构与功能将成为后基因组时代的一项重要任务。本文介绍了主要的蛋白质结构和序列数据库资源,在此基础上提出了一种高效的整合方法,并简述蛋白质结构与功能预测的基本方法、进展及其改进,展望了蛋白质预测技术的前景。     

Neuroprotective Function of Cellular Prion Protein in a Mouse Model of Amyotrophic Lateral Sclerosis

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1^G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1^G93APrp−/− mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1^G93APrp+/+ mice. Additionally, during disease progression, SOD1^G93APrp−/− mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1^G93APrp−/− mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1^G93APrp−/− versus SOD1^G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-κB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1^G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.Amyotrophic lateral sclerosis is characterized by rapid degeneration of motor neurons in the spinal cord, brain stem, and cortical Betz cells. As a result, focal muscle wasting, weakness, and spasticity develop focally. These symptoms ultimately lead to global paralysis. Patients usually die due to respiratory failure within 3 years of symptom onset.¹The causes of ALS are diverse; 10 to 15% of cases are familial with autosomal dominant inheritance, and 20% of these are related to point mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). SOD1 is a ubiquitously expressed homodimeric protein that catalyzes the reaction of O₂⁻ to O₂ and H₂O₂, which is then further metabolized by glutathione peroxidase. Mice overexpressing human mutated SOD1 (muSOD1) linked to ALS, develop disease resembling ALS in humans by a toxic gain of function.² Several properties of muSOD1 were proposed to contribute to toxic gain of function, including enhanced peroxidase activity and formation of peroxynitrite, changes in copper and zinc binding, and aggregation of the enzyme. ALS progression is accompanied by oxidative stress processes, glutamate-induced excitotoxicity, cytoskeletal abnormalities, inflammatory processes, and toxicity via extracellular muSOD1.^2,3 The apoptotic cascade is activated in the ALS model, shown by sequential activation of caspase-1 and −3.⁴ Interestingly, Bcl-2 overexpression had a neuroprotective effect and, like intrathecal administration of caspase inhibitors, led to a slowed disease progression and increased life span in these mice.^5,6 More recently, it was proposed that not only neurons themselves contribute to the neurodegenerative process in ALS but also non-neuronal cells, particularly microglia and astrocytes.^7–10 Expression of muSOD1 in non-neuronal cells was sufficient to induce cell death in nearby motor neurons lacking muSOD1.^11,12Since an early decrease of prion protein (Prp) mRNA has been described in an ALS model,¹³ loss of Prp function might contribute to the neurodegenerative process not only in prion diseases but also in ALS. A number of physiological functions have meanwhile been attributed to Prp, including antioxidative and anti-apoptotic properties and involvement in transmembrane signaling and cell adhesion.¹⁴ Additionally, as recently reported in cell culture models, Prp regulates astrocytic signaling^15,16 and thereby might also influence neuron−glia signaling relevant for ALS pathogenesis. Clarifying the role of Prp in models of neurodegeneration is of special interest, since Prp as membrane protein might become an easily accessible drug target for treatment of neurodegenerative diseases as a spin-off of the current search for antiprion drugs.¹⁷In the present study, we analyzed the function of physiological Prp in a mouse model of ALS by cross-breeding mice transgenic for human SOD1 with G93A mutation (SOD1^G93A) with Prp knockout (Prp−/−) mice. Characterization of the SOD1^G93APrp−/− mice concerning motoric properties, disease progression, and life span, paralleled by histopathological, immunochemical, and proteomic analyses, revealed that Prp has a protective role potentially by influencing mechanisms assuring neuronal survival.     

PrPC介导的细胞信号转导

朊病毒病的发生是由于细胞正常朊蛋白PrPC转变成了异常构象的PrPSc形式.PrPC 的生理学功能目前尚不完全明确,可能与铜离子代谢、脂质摄取以及细胞信号传递有关.PrPC可以与小窝蛋白相互作用而活化Fyn非受体酪氨酸激酶从而引起下游信号通路的转导;可以作为受体与PrPC键合多肽结合后激活cAMP/PKA信号通路;以及引起细胞内钙离子浓度变化而活化信号通路.     

体外循环对细胞免疫功能的影响及其主要影响因素分析

测定实验组(60例CPB心脏手术)、对照组(20例非CPB心脏手术)围手术期红细胞C3b受体花环率、红细胞免疫复合物花环率;T淋巴细胞亚群;白细胞、红细胞、淋巴细胞计数.进行各时点指标与术前比较的方差分析(x±s),组间两两比较配对t检验;进行不同氧合器、CPB时间、体重、年龄等多因素比较的方差分析.探讨CPB对机体细胞免疫功能的影响及其主要影响因素.结果:实验组红细胞、T淋巴细胞免疫功能及淋巴细胞和红细胞计数均较术前显著下降,自停CPB持续至术后3～8天.白细胞计数在停CPB时下降,术后3～8天高于术前水平.CPB对细胞免疫功能的损害作用与CPB时间、手术失血量呈正相关;与病人年龄、体重、CPB温度呈负相关.不同性别、病种组免疫功能损害程度亦有一定差异.膜式氧合器组较鼓泡式氧合器组细胞免疫功能损害轻.结论:CPB的气血界面、血细胞破坏及人工管道系统均可导致血液有形成分破坏,损害机体的细胞免疫功能.在围手术期监护中应予以重视.     

重组P16蛋白在大肠杆菌中的表达纯化及其功能研究

【摘 要】： 目的 在原核细胞中表达P16蛋白并纯化,研究外源性P16蛋白对肿瘤细胞周期的影响。方法 构建外源性P16融合蛋白的原核表达载体pQE31-p16。IPTG诱导大肠杆菌BL21菌株表达P16融合蛋白,通过亲和层析柱纯化目的蛋白,经Western-blot鉴定后, 将P16蛋白转导入人肺腺癌细胞系A549中, 利用免疫细胞化学方法对蛋白进行细胞内定位, 同时绘制细胞生长曲线,并用流式细胞仪分析细胞周期的变化。结果 所构建P16融合蛋白的原核表达载体pQE31-p16,可在大肠杆菌中稳定表达,通过纯化获得P16融合蛋白。利用lipofectamine2000转染试剂可将P16蛋白转入A549细胞中,生长曲线与流式细胞术结果显示P16蛋白可抑制肿瘤细胞增殖。结论 原核细胞表达的P16蛋白经纯化,转导进入A549细胞,可影响其生长周期,抑制A549细胞的增殖。     

PTP的结构与功能

蛋白质分子中酪氨酸残基的可逆性磷酸化作为真核生物信号转导的一个重要组成部分,参与了多种细胞功能调节,包括细胞增殖、迁移以及细胞间相互作用等。目前认为这种可逆性的磷酸化调节主要是受控于蛋白酪氨酸激酶(PTK)及蛋白酪氨酸磷酸酯酶(PTP)这两种酶活性的动态平衡。因此,与PTK一样,PTP对于体内各种生命活动起着非常重要的生物学作用。文章综述了近年来PTP在信号转导中的调控作用,特别是其在肿瘤发生、发展过程中的作用、以及其本身的结构与调控的研究进展。     

Immunoglgbulin Levels and Cellular Immune Function in Lead Exposed Workers

The immunological status of lead acid batters workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations ranging from safe to toxic levels has been examined and compared with those of non-exposed, age and sex matched controls. No differences in the serum concentrations of IgG, IgA and IgM between the populations were observed and there existed no correlation between blood lead level or ALA-U concentrations and serum immunoglobulin levels. In addition assessment was made of the capacity of peripheral blood mononuclear cells to respond to the mitogen phytohaemagglutin in (PHA), a correlate of T cell function. As before, there was no difference between exposed and control populations and no correlation between reactivity and blood lead concentration. Our data suggest that chronic exposure to lead fail to compromise lymphocyte function in man.