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1.
目的从细胞凋亡角度探讨不同剂量法舒地尔(fasudil)对升主动脉缩窄压力超负荷心力衰竭大鼠的影响及作用机制。方法采用升主动脉缩窄术建立大鼠心力衰竭模型。观察不同剂量fasudil治疗心力衰竭时对心肌细胞凋亡指数(AI)、bcl-2、c-myc蛋白表达水平的影响。结果fasudil干预心功能不全可以使心肌细胞凋亡指数降低,使bcl-2蛋白表达水平上调,c-myc蛋白表达水平降低,且剂量大时效果更明显。结论fasudil能有效减少心力衰竭大鼠心肌细胞凋亡指数,使bcl-2蛋白表达水平上调,c-myc蛋白表达水平降低,防治心力衰竭,这是其治疗心力衰竭的重要机制之一,且剂量大时更明显。  相似文献   

2.
PDCD5在类风湿关节炎成纤维样滑膜细胞凋亡中表达上调   总被引:8,自引:0,他引:8  
为了研究程序化细胞死亡因子5(PDCD5)在类风湿关节炎成纤维样滑膜细胞凋亡中的作用,在不同的时间加入有效剂量100 nmol/L雷公藤内醇酯(triptolide)后,采用实时定量PCR、RT-PCR、Western 印迹和直接免疫荧光染色方法检测体外分离培养的类风湿关节炎成纤维样滑膜细胞中PDCD5在mRNA和蛋白水平的表达及蛋白表达特征.在雷公藤内醇酯诱导类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5mRNA表达水平明显地渐次增加,呈现一种明确的时间依赖性递增表达模式,而PDCD5蛋白有时间依赖性表达上调持续16 h,并维持在相对恒定水平.直接免疫荧光染色结果显示,在正常体外培养的类风湿关节炎成纤维样滑膜细胞中,PDCD5蛋白的表达较弱,且主要分布在细胞浆.经雷公藤内醇酯处理4 h后,大多数细胞有PDCD5蛋白的聚集,直至12 h,细胞核周围PDCD5蛋白聚集显著增强.36 h后,PDCD5蛋白以核固缩的形式存在于凋亡的RA FLS中,细胞核染色质明显浓缩,片段化并出现了凋亡小体.上述结果表明,在类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5表达上调并在凋亡早期出现核转位,PDCD5蛋白核转位要早于凋亡小体形成.PDCD5蛋白核转位是类风湿关节炎成纤维样滑膜细胞凋亡的早期事件,PDCD5不仅参与了类风湿关节炎成纤维样滑膜细胞的凋亡过程,而且在类风湿关节炎滑膜增生的凋亡调节中起到重要调节作用.  相似文献   

3.
目的:通过5-Fu诱导携带有野生型p53基因的HCT116和携带有突变性p53基因的HT-29两种结肠癌细胞系,比较两株细胞在各时间点凋亡水平和PUMA mRNA表达情况的差异,探讨PUMA对细胞凋亡的作用及诱导其表达的基本途径。方法:用逆转录聚合酶链反应(RT—PCR)检测不同结肠癌细胞株HT-29、HCT116在抗肿瘤药物5-Fu作用下不同时间点结肠癌细胞株内PUMA mRNA表达水平的差异,用吖啶橙/溴化乙啶(AO/EB)荧光染色检测各时间点细胞的凋亡水平,分析其与PUMAmRNA表达水平之间的关系。结果:携带有野生型P53基因的结肠癌细胞株HCT116在5-Fu作用下6h即可出现PUMA mRNA的表达,随着药物作用时间的延长其表达强度增加,并且与细胞凋亡水平呈正相关;含有突变型P53基因的结肠癌细胞株HT-29在5-Fu作用下无PUMA的表达。结论:通过5-Fu诱导细胞凋亡出现的PUMA表达需要野生型P53基因,突变型P53基因无法诱导PUMA的表达。PUMA与结肠癌细胞凋亡水平呈正相关,是一种促凋亡蛋白。  相似文献   

4.
目的:探讨survivin在非小细胞肺癌组织(non small cell lung cancer,NSCLC)中的表达,及其与bcl2、p63蛋白表达的相关性。方法:应用二步法免疫组织化法,检测survivin、bcl-2、p63蛋白在60例NSCLC组织和20例正常肺组织中的表达。结果:肺癌组织中的survivin蛋白阳性率(56.67%)明显高于正常肺组织15%),有显著性差异;(p〈0.05)Ⅲ期surviving蛋白阳性表达率72.73%(16/22)明显高于Ⅰ+Ⅲ期survivin47.37%(18/38)。有显著差异;(p〈0.05)survivin蛋白表达与患者年龄、病理类型、组织分化程度,淋巴结转移情况无关(P〉0.05)NSCLC组织bc 1-2蛋白表达阳性、阴性组中,survivin蛋白阳性表达率分别为66.67%(18/27)和48.48%(16/33),两者比较,差异有显著性(P〈0.05);p63蛋白表达阳性、阴性组中,survivin蛋白阳性表达率分别为53-33%(16/30)和60%(18/30),两者比较,差异有显著性(P〈0.05)survivin,蛋白与bc1.2蛋白的表达呈正相关。survivin蛋白与p63蛋白的表达呈正相关。结论:survivin在NSCLC组织中表达上调,通过抑制细胞凋亡,在NSCLC的发生和发展中起到重要作用。survivin,bcl-2与p63它们分别在肺癌发生发展过程中不同途径上抑制肺癌细胞的凋亡,对肺癌早期诊断有一定的意义。对3种蛋白进行联合检测,更有利于肺癌的早期诊断和判断肺癌的分化程度、临床分期、淋巴结是否转移及病人的预后。survivin与bcl-2及survivin与p63可能起协同作用、并可能会成为NSCLC基因治疗的新靶点。  相似文献   

5.
运用定量显微形态分析技术、免疫荧光实验技术和MTT分析法,分别对裱衬纤维连接蛋白(fibronectin,FN)和应用肌球蛋白轻链激酶(myosin light chain kinase,MLCK)抑制剂ML-7作用下,肝细胞L02与肝癌细胞HepG2细胞骨架装配和Rho蛋白表达变化,以及细胞迁移能力进行检测和定量表征,了解肝癌细胞侵袭转移的胞内分子基础。结果显示:1)L02 Rho蛋白表达水平明显低于HepG2,裱衬FN低浓度(1~5 μg/mL)使 L02 Rho蛋白表达进一步下调,HepG2 Rho蛋白表达水平却明显增高,而裱衬 FN高浓度(10~40 μg/mL)L02Rho蛋白升高超过对照组,HepG2 Rho蛋白表达下降,且远低于对照组水平;2)随着裱衬FN浓度的增加,HepG2增殖抑制明显;3)裱衬低浓度FN使HepG2迁移运动能力增强,而高浓度FN使细胞净位移和迁移轨迹离散度减少;4)ML-7低浓度(6 μmol/L)使L02 Rho蛋白表达下调,而对HepG2 Rho蛋白表达无明显影响,L02细胞骨架解聚较HepG2明显;ML-7高浓度(10 μmol/L)HepG2Rho蛋白表达减少,且迁移速率降低;L02 Rho蛋白表达无进一步下调。说明:1)细胞粘附状态对调节HepG2 和L02Rho蛋白表达呈相反趋势;2)HepG2对骨架收缩抑制的响应较L02迟缓;3)HepG2Rho蛋白表达水平与其迁移能力呈正相关。  相似文献   

6.
间隙连接蛋白Cx43在人胚肺和肺癌细胞表达的研究   总被引:7,自引:0,他引:7  
细胞与细胞之间通过细胞膜上的间隙连接通道交换小分子和离子进行细胞间通讯,对细胞增殖分化调控和机体内环境稳定有重要作用。用间隙连接蛋白Cx43cDNA探针Northern印迹杂交,Cx43抗体免疫荧光染色和罗氏黄荧光染料传输方法检查,正常人胚肺细胞的Cx43在mRNA和蛋白水平有高表达,Cx43蛋白免疫荧光分布在间隙连接的部位,细胞间隙连接通讯功能明显。与正常相反,人肺癌PG系细胞Ck43无论在mRNA或蛋白质水平都无表达,细胞通讯功能缺陷。结果表明Cx43在培养的人胚肺细胞有功能性表达。人肺癌PG细胞通讯功能缺陷与Cx43基因转录抑制有关。对Cx基因的抑癌基因性质进行讨论。  相似文献   

7.
《植物杂志》2010,(8):6-6
近日,科学家开发了一项能够在成体水平小鼠脑区的复杂神经网络系统中特异性标记单个神经元细胞及其神经突触分布形式的技术。该项技术利用转基因方法在小鼠不同脑区的神经元细胞可控地表达不同的荧光蛋白和突触囊泡蛋白,应用不同分子标记对神经元细胞及其突触进行特异性标记,能够在单个神经元细胞水平上提供详尽的三维神经突触分布信息,  相似文献   

8.
目的:研究桩蛋白(Paxillin,Pxn)在胰腺发育不同阶段的表达和细胞定位.方法:运用RT-PCR技术检测Pxn在大鼠胰腺发育不同阶段的mRNA表达水平;运用免疫组织化学检测不同时期桩蛋白在胰腺的定位.结果:RT-PCR结果显示Pxn的mRNA表达量胚胎期高于新生和成年期;免疫组织化学结果显示在不同发育时期桩蛋白不仅在外分泌胰腺有表达,而且在胰岛也有表达.结论:具有调节细胞聚集、粘附迁移功能的桩蛋白可能参与出生后胰岛重塑.  相似文献   

9.
将萜烯类吲哚生物碱代谢关键酶--色氨酸脱羧酶(TDC)的编码基因转到烟草(Nicotiana tabacum L.)植物体内,标定在不同的亚细胞区室表达。通过蛋白免疫印迹法和色胺在植物体内的累积量测定分析,对转基因植物进行筛选。结果表明,TDC在叶绿体和胞液中高效表达,TDC在叶绿体中的表达水平最高,高于在胞液中的表达,在内质网和液泡中表达水平很低,用蛋白免疫印迹法未检出。  相似文献   

10.
组织转谷酰胺酶(transglutaminase 2, TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关。有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道。为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除的MDCK稳定细胞系,感染H1N1亚型流感病毒48 h后检测病毒NP和NS1蛋白mRNA和蛋白表达水平的变化,测定病毒滴度。结果显示,过表达TGM2能够有效抑制H1N1亚型流感病毒的NP、NS1基因的表达,而敲除TGM2可上调病毒NP、NS1基因的表达,其中NP蛋白的蛋白表达情况与mRNA水平一致。病毒增殖曲线结果显示,TGM2过表达后H1N1亚型流感病毒滴度显著降低,而敲除TGM2后结果相反,48h时病毒滴度在敲除细胞中达到高峰,进一步证明TGM2在MDCK细胞中参与对H1N1亚型流感病毒增殖的抑制。通过对流感病毒应答信号途径下游基因的表达分析发现,TGM2可能通过促进激活JAK-STAT分子途径和抑制RIG-1信号途径,抑制H1N1亚型流感病毒增殖。以上发现对于揭示...  相似文献   

11.
重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素   总被引:8,自引:0,他引:8  
高效表达重组蛋白 ,对于生物制药意义重大。大多数药用蛋白是糖蛋白 ,中国仓鼠卵巢细胞 (Chinesehamsterovarycell,CHO)是目前重组糖基蛋白生产的首选体系。影响外源蛋白在CHO细胞中表达的因素很多 ,从CHO细胞表达体系、表达载体系统、外源基因、表达细胞株的加压扩增与筛选、细胞大规模培养等方面对CHO高效表达加以阐述 ,同时提出存在的问题和未来的发展方向。  相似文献   

12.
无细胞蛋白质合成(cell-free protein synthesis,CFPS)是一种在体外快速合成目标蛋白质的方法,通过构建含有CFPS系统的人造细胞,能够实现蛋白质的高通量表达和功能性膜蛋白的体外重构.本文详细综述了4种CFPS系统(包括大肠杆菌裂解液、兔网织红细胞裂解液、小麦胚芽提取物、酵母提取物)的适用范围和优缺点,总结了基于CFPS系统构建的人造细胞体系内蛋白质合成的研究现状,以及该领域面临的挑战及未来的发展方向.  相似文献   

13.
Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal‐cell biotechnology. Difficult‐to‐express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full‐length protein constructs. Post‐translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time‐, labor‐, and cost‐intensive. It is clearly desirable to find an automated and miniaturized fast multi‐sample screening method for protein expression in such systems. With this in mind, in this paper a high‐throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide‐binding Oligomerization Domain‐containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid‐based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975–1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.  相似文献   

14.
任何一个蛋白质合成系统的最终目标都是表达所需要的蛋白质(目的蛋白质)而不产生其他的细胞蛋白质。目前,只有两种方法可以成功的表达目的蛋白质:体外无细胞蛋白质合成系统和体内单蛋白生产(SPP)系统。综述现今不同的单蛋白生产系统,讨论他们的应用、优点和缺点。  相似文献   

15.
外源蛋白在中国仓鼠卵巢细胞中高效表达的策略   总被引:10,自引:0,他引:10  
高效表达外源蛋白,在生物制药中有重要意义.中国仓鼠卵巢细胞(Chinese hamster ovary cell)是表达外源蛋白的最佳真核表达系统之一.影响外源蛋白在CHO细胞表达的因素甚多,主要包括载体、宿主细胞和外源基因几方面.深入了解和灵活运用它们之间的关系,有助于获得外源基因在CHO细胞中的高效表达.  相似文献   

16.
17.
Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.  相似文献   

18.
Systems for multigene delivery in mammalian cells, particularly in the context of genome engineering, have gained a lot of attention in biomolecular research and medicine. Initially these methods were based on RNA polymerase II promoters and were used for the production of protein complexes and for applications in cell biology such as reprogramming of somatic cells to stem cells. Emerging technologies such as CRISPR/Cas9-based genome engineering, which enable any alteration at the genomic level of an organism, require additional elements including U6-driven expression cassettes for RNA expression and homology constructs for designed genome modifications. For these applications, systems with high DNA capacity, flexibility and transfer rates are needed. In this article, we briefly give an update on some of recent strategies that facilitate multigene assembly and delivery into mammalian cells. Also, we review applications in various fields of biology that rely on multigene delivery systems.  相似文献   

19.
It has been proposed that cyclical gene expression occurs at a large number of different times during the cell cycle. The existence of a large number of cycle-specific variations in mRNA and protein during the eukaryotic cell cycle raises the problem of how cell-cycle variations are regulated. This is the “infinite regression” or Russian Doll problem where postulating a cell-cycle specific control element pushes the explanation of cell-cycle variation back one step to the problem of how that control element varies during the cell cycle.PCR studies on unperturbed cells indicate Cyclin mRNA content is invariant during the cell cycle. Furthermore, calculations reveal that variations in mRNA content do not account for observed protein variations.Continuous and constant gene expression during the cell cycle, continuous protein accumulation, and protein breakdown only within the mitotic window solves the Russian Doll problem or infinite regression problem. These results, and theoretical ideas support an alternative view of the cell cycle where many of the proposed control systems do not exist.  相似文献   

20.
《朊病毒》2013,7(2):142-146
Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.  相似文献   

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