细胞程序式死亡途径的新进展

关于细胞程序式死亡(programmed cell death,PCD)的分类目前有多种不同的观点,如有凋亡、副凋亡、胀亡和自噬等.但随着对PCD的深入研究,发现过去的分类是不合理的.出现了彼此重叠的机制.这是由于研究者认识的局限性造成的.因此,很有必要对细胞程序式死亡途径进行重新认识.根据最新研究成果将PCD主要划分为7种,它们分别为经典凋亡、自体吞噬、凋亡样程序性死亡、坏死样程序性死亡、类凋亡、丝裂灾变和衰老.     

细胞程序性死亡的非Caspase依赖型途径

程序性细胞死亡是一种程序化的主动性细胞死亡,半胱胺酸天冬氨酸特异性蛋白酶家族(在该过程中起着不可忽视的作用.基于Caspase在程序性细胞死亡过程中所起的作用,将程序性细胞死亡分为两大类:Caspase依赖型和Caspase非依赖型.前者即典型的凋亡,后者包括自体吞噬、副凋亡、有丝分裂灾变、凋亡样程序性死亡、坏死样程序性死亡等.这些Caspase非依赖型的细胞程序性死亡途径与生理及病理现象密切相关.     

胱冬肽酶非依赖性的细胞程序性死亡

在多细胞有机体的组织内稳态维持和正常发育过程中,细胞程序性死亡发挥着重要的作用。细胞程序性死亡有多种形式(如细胞凋亡、类细胞凋亡和类坏死等),其中了解较清楚的是细胞凋亡。一直以来,胱冬肽酶(caspase)被认为是细胞凋亡发生中关键的一种蛋白酶。但是最近的研究表明,包括细胞凋亡在内的一些细胞程序性死亡可以以一种不依赖胱冬肽酶的方式发生。细胞程序性死亡与胱冬肽酶之间存在非依赖性关系。     

神经酰胺以Caspase依赖和非依赖方式诱导线粒体凋亡蛋白释放

本研究探讨了外源性C2-神经酰胺诱导入结肠癌HT-29细胞凋亡中,线粒体膜间隙凋亡蛋白的释放机制．不同浓度C2-神经酰胺作用HT-29细胞,流式细胞仪检测线粒体膜电位（△ψm）,线粒体／细胞液分离试剂盒分离亚细胞成分,聚丙烯酰胺凝胶电泳检测细胞色素C（Cytc）、高温必需蛋白A2（HtrA2）、线粒体源性半胱天冬氨酸蛋白酶第二活化因子（Smac）、凋亡抑制蛋白（XtAP）和半胱天冬氨酸蛋白酶-3（Caspase-3）蛋白表达水平．实验结果显示25和50μmol／L C2-神经酰胺作用细胞6h,△ψm即开始下降（P〈0．05）,且环孢霉素能通过调节线粒体膜通透性转换孔抑制△ψm的下降．C2-神经酰胺对Cyt c,HtrA2和Smac总蛋白表达没有明显影响,但能诱导Cyt c,HtrA2和Smac从线粒体释放入细胞液中,并下调XIAP蛋白的表达及活化Caspase-3．在Caspase抑制剂存在下,C2-神经酰胺仍能诱导Cyt c和HtrA2从线粒体释放,但不能诱导Smac释放．因此认为C2-神经酰胺能通过线粒体凋亡通路诱导HT-29细胞凋亡,C2-神经酰胺诱导Cytc和HtrA2从线粒体的释放是Caspase非依赖性的,而Smac释放是Caspase依赖性的．     

一种高效抑制Jurkat细胞表达RhoGDI2的方法

目的:RhoGDI2是RhoGTP酶的解离抑制因子,在白血病细胞内表达量很高。构建Rho GDI2短发夹RNA(sh RNA)的慢病毒载体,并鉴定该慢病毒在急性T细胞白血病细胞系Jurkat内的抑制效率。方法:针对人rho GDI2基因序列合成sh RNA序列,通过限制性内切酶Bam HⅠ和XhoⅠ、T4DNA连接酶,将该sh RNA序列插入慢病毒载体p EN_h H1c,测序正确后通过LR重组酶与p DSL_hp UGIP重组,转化感受态细菌,筛选阳性克隆,测序鉴定正确后用脂质体法将质粒与ps PAX2、p MD2共转染293T细胞、包装病毒并侵染Jurkat细胞,采用荧光显微镜观察侵染效率、West-ern印迹鉴定Rho GDI2 sh RNA在Jurkat细胞内的表达。结果:构建的Rho GDI2 sh RNA慢病毒成功侵染Jurkat细胞并抑制Rho GDI2的表达。结论:构建并鉴定了RhoGDI2 shRNA的慢病毒表达质粒,为研究RhoGDI2在白血病中的作用奠定了基础。     

神经细胞死亡的细胞和分子机制

神经细胞死亡的细胞和分子机制朱国璋,张永莲,龚岳亭（中国科学院上海生物化学研究所,上海２０００３１）关键词神经细胞死亡,细胞和分子机制无论是在脊椎动物或无脊椎动物的神经系统发育和神经网络构建过程中,在外周神经和中枢神经细胞增殖、分化、成熟的同时,伴随...     

白藜芦醇通过诱导细胞自噬性死亡抑制宫颈癌的研究

白藜芦醇作为一种广泛存在于药食同源植物中的非黄酮类多酚化合物,其抗肿瘤效果受到广泛关注,但在抑制宫颈癌方面仍缺乏体内效应的实验依据.本研究通过体内实验发现白藜芦醇具有明显的抗肿瘤生长作用,组织水平LC3B、P62和Beclin-1表达改变,推测白藜芦醇可能通过促进癌细胞的过度自噬抑制宫颈癌的进展;进一步通过体外细胞实验...     

应用原子力显微镜分析正常淋巴细胞和Jurkat细胞的形态和机械性质

淋巴细胞形态和机械性质的变化与人的健康、疾病的治疗和诊断有着密切关系。本研究利用原子力显微镜研究淋巴细胞和Jurkat细胞形态和机械性质。结果显示,这2种细胞的形态较为相似,但通过对力曲线的分析得出这2种细胞的机械性质明显不同。正常淋巴细胞粘弹力范围大致为(796.7±248.5)pN,而Jurkat细胞分布于(158.5±37.5)pN;正常淋巴细胞的杨氏模量(0.471kPa±0.081kPa)近4倍于Jurkat细胞(0.0964kPa±0.0229kPa);而Jurkat细胞(4.322mN/m±0.382mN/m)的硬度近2倍于正常淋巴细胞(2.278mN/m±0.488mN/m)。结果表明原子力显微镜能可在临床诊断上区分正常细胞与肿瘤细胞,即使两者形态区别不明显。     

p53转录非依赖活性介导细胞凋亡

p53主要通过两条途径诱导细胞凋亡：p53作为转录因子,促进细胞凋亡的靶基因的表达上调,如PUMA、NOXA、PIDD、p53AIP1、COP1等,并通过这些蛋白参与内源和外源凋亡途径;另一方面,胞浆中的p53能转位到线粒体,激活内源性的线粒体途径,促进凋亡。后者已成为研究p53促凋亡机制的热点。本文就p53对转录非依赖活性诱导细胞凋亡途径的研究进展作一概述。     

Naegleria fowleri Induces Jurkat T Cell Death via O-deGlcNAcylation

The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri–induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.     

去甲斑蝥素对白血病Jurkat细胞增殖的影响

目的:观察不同浓度去甲斑蝥素(NCTD)对急性T淋巴细胞白血病细胞株Jurkat增殖的影响。方法:体外培养的Jurkat细胞,用0、5、10、20、40 mg/LNCTD作用6、12、24、48、72 h后,通过倒置显微镜观察细胞形态、密度变化,MTT法检测细胞增殖抑制率,确定其最佳作用浓度及时间。然后将细胞分为NCTD组、长春新碱组、NCTD+长春新碱组,作用24、48、72 h后观察细胞形态、密度变化,MTT法检测细胞增殖抑制率。结果:倒置显微镜显示:随NCTD浓度增加、作用时间延长,细胞形态不规则,胀大、固缩,分布稀疏,大量死亡;MTT法显示:随NCTD浓度增加、作用时间延长,细胞增殖抑制率渐升高,20 mg/L作用72 h时最高(59.24%)。NCTD组与长春新碱组比较差异无统计学意义(P0.05),NCTD+长春新碱组抑制率(77.40%)明显高于NCTD组,差异有统计学意义(P0.05)。结论:NCTD可以浓度和时间依赖性方式抑制Jurkat细胞的增殖,与长春新碱联合后作用增强。     

A Novel Apoptosis-like Pathway, Independent of Mitochondria and Caspases, Induced by Curcumin in Human Lymphoblastoid T (Jurkat) Cells

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.     

Ridaifen-G诱导细胞死亡不依赖于细胞凋亡蛋白酶(英文)

通过对Ridaifen-G(RID-G)诱导造血细胞U937、Raji、THP-1和IM-9死亡是否需要Z-VAD-fmk(一种细胞凋亡蛋白酶抑制剂)的研究,发现RID-G以细胞凋亡蛋白酶非依存性的方式诱导细胞死亡,并伴随有线粒体功能紊乱。Z-VAD-fmk对U937细胞的死亡没有影响,但抑制etoposide诱导的细胞凋亡;DNA片段化结果表明,RID-G可破坏Raji和THP-1细胞的DNA,经RID-G处理后的U937细胞的DNA条带没有经etoposide处理的清晰。此外,Z-VAD-fmk对U937、THP-1和Raji细胞DNA的片段化程度有不同的影响,抑制细胞死亡。这些结果表明,RID-G诱导的非典型细胞死亡不依赖于caspase,且伴随有线粒体功能紊乱。     

Programmed Cell Death 6 (PDCD6) Protein Interacts with Death-Associated Protein Kinase 1 (DAPk1): Additive Effect on Apoptosis via Caspase-3 Dependent Pathway

Programmed cell death 6 (PDCD6) protein is a 22 kDa EF-hand type Ca²⁺-binding protein involved in apoptosis. To define the regulating mechanism of PDCD6 activity in the apoptotic pathway, we searched a human ovary cDNA library for a novel PDCD6 binding protein using a yeast two-hybrid system. The selected protein was the human death-associated protein kinase 1 (DAPk1), another protein that functions as a positive mediator of apoptosis. Co-transfection of PDCD6 and DAPk1 cDNA into a tumor cell line accelerated apoptosis via caspase-3 dependent pathway.J.H. Lee and S.B. Rho contributed equally to this workRevisions requested 4 March 2005; Revisions received 10 May 2005     

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death

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Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.