鞘脂与细胞凋亡

随着生物技术的不断发展,近年来对鞘脂类物质的研究不断深入。鞘脂质除了在细胞骨架的迁移、血管发生、胚胎发育和信号转导等方面起重要作用外,最近的研究发现鞘脂及其代谢物(神经酰胺、鞘氨醇、鞘氨醇-1-磷酸)能诱导多种肿瘤和恶性增殖细胞(如腺癌、结肠癌、肝肿瘤、肺癌、鼻咽癌等)的凋亡。本文着重对鞘脂与细胞凋亡相关的最新研究进展进行综述。     

神经酰胺与细胞凋亡

细胞凋亡 (apoptosis) 又称细胞的程序性死亡,参与机体许多生理和病理过程.近年来的研究表明细胞凋亡与鞘脂质代谢有密切的关系,鞘磷脂的水解产物神经酰胺作为脂质第二信使在诱导和抑制细胞凋亡中起着重要的作用.     

神经酰胺与细胞凋亡

神经酰胺与细胞凋亡叶珉王亚新（上海第二医科大学附属瑞金医院血液学研究所,上海２０００２５）关键词神经酰胺细胞凋亡信号转导细胞凋亡为维持机体内环境稳定所必需,它与有丝分裂相互协调,共同调控胚胎发育、器官的发育和退化、免疫和造血等功能。研究发现,神经酰胺...     

三氧化二砷诱导细胞凋亡对线粒体的影响

线粒体作为细胞能量的“动力工厂”,在细胞的生理过程中起着重要的作用,本文采用三氧化二砷（Ａｓ２ Ｏ３） 诱导ＭＧＣ－ ８０３ 细胞凋亡,通过罗丹明１２３ 和Ｈｏｅｃｈｅｓｔ３３２５８ 对细胞进行荧光标记,利用具有光子计数成像能力的超高灵敏度荧光显微镜观察凋亡细胞与未加入Ａｓ２ Ｏ３ 的对照组细胞的区别,发现Ａｓ２ Ｏ３ 在诱导细胞凋亡时可引起线粒体内膜电位差减小     

线粒体在细胞凋亡中的作用

线粒体在凋亡中的作用越来越受到重视,它在细胞凋亡中起中心作用,释放凋亡活性物质,介导凋亡的酶促反应,参与凋亡调控,决定细胞是凋亡还是坏死。     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

紫外照射神经酰胺的生成及神经酰胺引起的细胞凋亡

参照文献报道的方法建立了测定细胞神经酰胺的激酶催化法,以该法所做的标准曲线在一定浓度范围内有很好的线性关系。以ＵＶＢ照射ＮＩＨ ３Ｔ３细胞,可以使细胞神经酰胺的水平在１分钟之内迅速升高１．５倍以上,基本上达到了细胞所能生成神经酰胺的最高峰值。可细胞膜的外源性神经酰胺（Ｃ２－ｃｅｒａｍｉｄｅ）作用于ＮＩＨ ３Ｔ３细胞后能够导致细胞的凋亡和坏死,其凋亡率在一定剂量范围内随神经酰胺浓度的增加而增加;而在     

线粒体与细胞凋亡

细胞凋亡是一种由基因控制的自主性死亡过程。近年来研究发现,线拉体在细胞凋亡过程中起重要作用,它可以通过改变膜通透性、释放凋亡活性物质等介导细胞凋亡。     

梓醇对氧糖剥夺诱导PC1 2 细胞凋亡的保护作用

目的：观察梓醇对氧糖剥夺（OGD）诱导PC12细胞凋亡的保护作用。方法：采用Hoechst 33258 DNA染色法,四甲基偶氮唑盐（MTT）检测细胞活性;化学比色法测定乳酸脱氢酶（LDH）的释放量,用流式细胞技术检测细胞凋亡比例以及P53和Bcl-2蛋白。结果：OGD可导致PC12细胞活力明显下降,LDH释放量增加、P53蛋白表达上升,Bcl-2蛋白表达下降。梓醇可明显改善细胞形态结构,显著降低LDH释放量、降低P53蛋白的表达,提高Bcl-2蛋白的表达,降低细胞凋亡率。结论：梓醇通过调节细胞凋亡相关基因的表达而抑制细胞凋亡。     

猪丁型冠状病毒诱导的细胞线粒体凋亡

猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)是一种新型的猪肠道致病性冠状病毒,可引起猪群剧烈腹泻及呕吐,但致病机制尚不清楚。本研究检测了PDCoV感染诱导的细胞凋亡。Caspase酶活性检测显示,在PDCoV感染的细胞中,caspase 3、caspase 8和caspase 9的活性随病毒感染量的增多而显著提高,类似的现象未能在紫外灭活病毒感染的细胞中观察到,表明PDCoV感染可同时激活内源性与外源性细胞凋亡通路,并暗示细胞凋亡的诱导依赖于病毒复制。为深入探究PDCoV诱导的内源性细胞凋亡,分别检测胞浆和线粒体中细胞色素C与凋亡诱导因子。结果显示,与正常细胞相比,PDCoV感染细胞从线粒体释放到胞浆的细胞色素C显著增多,且其释放量随着感染时间的延长而增多,而凋亡诱导因子始终定位于线粒体,提示PDCoV感染通过促使线粒体膜间隙的细胞色素C进入胞浆而启动caspase依赖的线粒体凋亡通路。本研究初步揭示了PDCoV诱导细胞凋亡的机制。     

Mitochondrial Ceramide and the Induction of Apoptosis

In most cell types, a key event in apoptosis is the release of proapoptotic intermembrane space proteins from mitochondria to the cytoplasm. In general, it is the release of these intermembrane space proteins that is responsible for the activation of caspases and DNases that are responsible for the execution of apoptosis. The mechanism for the increased permeability of the mitochondrial outer membrane during the induction phase of apoptosis is currently unknown and highly debated. This review will focus on one such proposed mechanism, namely, the formation of ceramide channels in the mitochondrial outer membrane. Ceramides are known to play a major regulatory role in apoptosis by inducing the release of proapoptotic proteins from the mitochondria. As mitochondria are known to contain the enzymes responsible for the synthesis and hydrolysis of ceramide, there exists a mechanism for regulating the level of ceramide in mitochondria. In addition, mitochondrial ceramide levels have been shown to be elevated prior to the induction phase of apoptosis. Ceramide has been shown to form large protein permeable channels in planar phospholipid and mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway with which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Ceramide in Apoptosis: A Revisited Role

The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed.     

Ceramide Glycanase Activities in Human Cancer Cells

Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGases, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhibit the enzyme activity substantially in both tumor cells lines. The mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase proteins.Radiolabeled substrates, labeled at the sphingosine double bond or at the 3-position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme preparations isolated from different cancer cells (apparent Km, 500 M for nLcOse5[³H-DT]Cer and 350 M for GgOse4[sph-3-³H]Cer with Colo-205 enzyme). Structural analogues of ceramide and sphingosine (L-PPMP, L-PDMP, alkylamines, and Tamoxifen) inhibited cancer cell CGase activities in vitro.     

Sphingosine Forms Channels in Membranes That Differ Greatly from Those Formed by Ceramide

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.     

Induction of Oligodendrocyte Apoptosis by C2-Ceramide

Tumor necrosis factor- induces oligodendrocytes apoptosis, and is known to stimulate the hydrolysis of sphingomyelin to form the lipid mediator, ceramide. These data encouraged us to determine whether ceramide itself is able to induce apoptosis in oligodendrocytes. For this purpose the cell-permeable ceramide analog, C₂-ceramide was used. Treatment of bovine oligodendrocyte cell cultures with this compound induced cell death in a time- and concentration-dependent manner. The induction of cell death was specifically associated with the action of C₂-ceramide and could not be elicited by dioctanoylglycerol (DC₈) or phorbol 12-myristate 13-acetate (PMA). Treatment of the cultures with neutral sphingomyelinase, which increased the hydrolyses of endogenous sphingomyelin, resulted in oligodendrocyte death, whereas exposure of the cells to phospholipase C and A₂ did not. C₂-ceramide treatment caused DNA fragmentation. Morphologic analysis of the cells showed that C₂-ceramide treatment resulted in a loss of their processes, reduction of cell volume, chromatin condensation, and formation of apoptotic bodies. These results indicate that ceramide can induce oligodendrocyte apoptosis, and suggest that sphingolipid metabolism plays a key role in the regulation of this process.     

CrmA Protects Against Apoptosis and Ceramide Formation in PC12 Cells

TNF- activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF- caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF--induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3–mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD-CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis.     

酸性鞘磷脂酶/神经酰胺通路在疾病发生发展中 作用机制的研究进展

酸性鞘磷脂酶/神经酰胺通路可介导细胞凋亡、炎症和自噬等多种细胞活动,与心脑血管疾病、代谢类疾病、肺部和肝部疾病以及 神经系统疾病等多种疾病的发生、发展密切相关。酸性鞘磷脂酶现已成为多种疾病的临床生物标记物和潜在的治疗靶点。综述酸性鞘磷脂 酶/神经酰胺通路在各种疾病中的生物学功能和作用机制最新研究进展,旨在为相关疾病的治疗提供新思路。     

线粒体膜间隙蛋白在细胞凋亡中的作用

线粒体除了作为细胞内的“能量工厂”外,在控制细胞凋亡中起主导作用。细胞凋亡时,线粒体膜通透性增加,释放可溶性线粒体膜间隙蛋白质,进一步破坏细胞结构。在这些致死性蛋白质中,有些(cytc、Smac／DIABLO、Omi／HtrA2等)能够激活caspases,另一些(endo G、AIF、Omi/HtrA2等)则以非caspase依赖的方式发挥作用。多种线粒体因子参与细胞凋亡,强化了细胞器在凋亡控制中的核心作用。     

神经酰胺与细胞凋亡

在多细胞有机体中,凋亡对正常组织的发育和稳态是必需的。神经酰胺（ceramide,Cer）作为一种脂类分子,不仅是细胞膜的重要组成部分,而且是重要的凋亡参与者。在细胞膜上,它能够自发地聚集,形成招募死亡受体和配体的结构域从而促进信号的传导和放大;在细胞质中,它作为信号通路的中介者,通过许多不同的分子机制去调控机体的凋亡通路;在细胞核膜和核内,通过其代谢变化,决定细胞的增殖或凋亡;而且它的浓度和位置以及与其它分子的比例及其相互作用都会影响到细胞和组织的命运。综述了近来关于Cer及其代谢物的凋亡作用。