细胞凋亡的信号通路

细胞凋亡是细胞程序性死亡的一种方式,与自噬和坏死有明显的区别。细胞凋亡的信号途径比较复杂,在凋亡诱导因子的刺激下经历不同的信号途径。本文就细胞凋亡的三条信号通路——线粒体途径、内质网途径和死亡受体途径做一综述,以便为人们进一步了解细胞凋亡发生的机制,从而对癌症及其他一些相关疾病的治疗奠定基础。     

猪MII期卵母细胞玻璃化冷冻后的凋亡途径研究

为阐明猪卵母细胞玻璃化冷冻后的细胞凋亡模式,本试验利用原位荧光染色技术对冻后卵母细胞死亡受体介导的外源性凋亡途径和线粒体介导的内源性凋亡途径中的Caspase 3、Caspase 8、Caspase 9和总Caspase活性进行检测,用RT-PCR技术对不同途径中关键基因进行mRNA表达检测。结果显示:猪MII期卵母细胞冷冻后,死亡受体外源性凋亡途径的Caspase 8荧光强度值(32.03)和线粒体内源性凋亡途径的Caspase 9荧光强度值(16.56),以及两者共同途径中的Caspase 3和总Caspase荧光强度值(16.70和8.43)均显著高于新鲜卵母细胞对照组所对应的荧光强度值(分别为4.02、4.83、4.23和3.08)。死亡受体外源性凋亡途径TNFα、FasL、CASP8和CASP3基因表达量也均有一定水平的提高,线粒体介导的内源性凋亡途径中CASP9、CASP3和P53基因表达水平呈上升趋势,而Bcl2、BAX、SOD1和survivin的基因表达水平显著下降。综上所述,死亡受体介导的外源性凋亡和线粒体介导的内源性凋亡共同参与了猪MII期卵母细胞冻后的凋亡过程。     

昆虫细胞凋亡的线粒体通路研究进展

介绍了哺乳动物和一些代表昆虫的线粒体凋亡途径研究进展,特别是与线粒体途径密切相关的凋亡调控因子,如细胞色素C,caspases家族,Bcl-2家族等的功能作用,阐述了线粒体在昆虫细胞凋亡中的作用,为进一步研究昆虫细胞凋亡机制以及发展以昆虫细胞凋亡因子为靶标的特异性杀虫剂提供理论基础。     

苦参素对D-氨基半乳糖苷诱导的肝细胞凋亡的干预研究

[目的]研究氧化苦参碱对肝细胞凋亡的影响。[方法]采用D-氨基半乳糖苷(D-GalN)诱导人源HL-7702肝细胞发生凋亡,探索最佳的凋亡诱导参数,建立肝细胞凋亡模型,并运用建立的体外肝细胞凋亡模型,探讨氧化苦参碱的抗凋亡作用。[结果]D-GalN在60mmol/L浓度下与HL-7702肝细胞孵育8 h为最优建模条件,在此处理条件下经凝胶电泳可见细胞凋亡特征性的DNA ladder;且与对照组相比,HL-7702细胞处理后其ALT、AST、SOD、MDA、Caspases-3水平均显著升高(P0.05)。[结论]D-GalN诱导的HL-7702肝细胞凋亡的发生机制可能与线粒体信号转导途径有关,而氧化苦参碱对D-GalN引起的肝细胞凋亡具有较强的干预作用,其抗凋亡机制可能与抑制Caspases-3、CytC、TNFα和TGF-β1等凋亡因子和诱导抗凋亡因子HO-1表达有关。     

线粒体途径参与FTY720诱导白血病细胞凋亡的研究

为研究FTY720诱导K562细胞凋亡与线粒体途径的关系。体外培养人白血病K562细胞,用不同浓度FTY720处理细胞24 h,台盼蓝染色法检测K562细胞存活率;流式细胞术分析细胞凋亡和线粒体功能。免疫印迹技术检测线粒体相关蛋白Bcl-2和Bax的表达。收集FTY720处理48 h细胞,流式细胞技术检测细胞周期。结果表明:随FTY720浓度的增加,K562细胞的存活率下降,细胞凋亡增加,线粒体膜电位下降,促凋亡蛋白Bax表达明显增加。细胞周期分析发现,Sub G1期细胞增加,而S期和G2M期细胞减少。表明线粒体途径参与FTY720诱导K562细胞凋亡。     

细胞凋亡对宰后肌肉嫩化作用机理的研究进展

嫩度是决定肉食用品质的重要指标。宰后肉的嫩度发生不连续变化,严重降低了消费者的购买意愿,因此阐明宰后嫩化机理一直是肉品科学领域的研究热点。自“凋亡”的概念引入至宰后肌肉嫩化过程后一直广受关注,动物被屠宰放血后,活性氧（reactive oxygen species,ROS）大量累积,ATP（adenosine triphosphate）逐渐耗尽,必然导致细胞死亡。宰后肌细胞死亡和肌肉嫩化都是在一系列调控因子作用下激活肌肉内源酶,并由内源酶水解蛋白质破坏细胞结构,因此这两个生化过程被认为高度相关。本文综述了宰后肌细胞主要以凋亡的形式死亡,分析了除凋亡外,宰后早期产生少量ROS时细胞会通过自噬启动自身防御系统,宰后后期ATP逐渐耗尽肌细胞可能从凋亡转变为坏死;明确了线粒体通路是宰后肌肉中细胞凋亡酶激活的关键路径,线粒体死亡因子释放是细胞内死亡级联反应的总开关,其开放状态直接决定着细胞以何种途径进行死亡,并进一步从线粒体膜通透化和内膜嵴重构两方面,讨论了宰后线粒体损伤诱导凋亡因子的释放机理;综述了线粒体损伤变化及其对嫩化过程的影响,并从线粒体通过参与能量代谢影响肌肉pH以及通过释放凋亡因子调控细胞凋亡酶活性两方面分析了其潜在机理;探讨了宰后肌肉线粒体与内质网间相互作用以影响Ca²⁺信号传导以及细胞凋亡过程,或与溶酶体相互作用,破坏溶酶体膜稳定性,使其释放组织蛋白酶以激活线粒体Bax和Bid而加速线粒体膜通透性;综述了细胞凋亡酶在宰后早期被激活,并参与部分肌原纤维蛋白的有限降解,但随着宰后时间的延长,ATP逐渐耗尽等因素导致细胞凋亡酶失活,因此细胞凋亡酶只参与宰后早期的嫩化过程。综述内容可为完善宰后肌肉嫩化过程提供理论参考。     

亚硒酸钠对黄曲霉毒素B_1致雏鸡胸腺细胞死亡受体通路活化的缓解作用研究

【目的】探究硒对AFB1致胸腺过度凋亡的缓解机制。【方法】180只1日龄科宝肉鸡随机分为4组,分别饲以对照日粮、AFB1日粮(0.6 mg/kg AFB1)、+Se日粮(0.4 mg/kg Se)和AFB1+Se日粮(0.6 mg/kg AFB1+0.4 mg/kg Se)21 d。【结果】添加硒可缓解AFB1所致的胸腺形态学损伤,适当缓解AFB1导致的T淋巴细胞亚群比例下降和凋亡率升高的现象;与AFB1组相比,AFB1+Se组胸腺TNF-α、caspase-8、caspase-3、caspase-9和JNK1的表达量降低,Bcl-2和t Bid的表达量升高。【结论】添加硒可缓解AFB1致雏鸡胸腺细胞凋亡率升高的机理与死亡受体通路中Fas-Fas L-FADD-caspase8-caspase3途径和Fas-ASK1-JNK-Bcl-2途径受到干预有关。     

细胞凋亡与细胞色素C

细胞凋亡是动植物最基本的生命活动,是一个有一系列酶参与的,并且由基因控制的主动的、高度有序的死亡过程。线粒体除了为细胞提供能量外,在细胞凋亡中也起着中心调控作用。研究发现,线粒体释放的细胞色素C是细胞凋亡过程的关键因素,已是近些年研究的热点。本文就细胞色素C从线粒体释放的机制及在凋亡中的作用进行综述。     

热应激对猪小肠组织形态和细胞凋亡的影响

采用HE染色观察猪小肠形态学变化,经PCR对猪小肠细胞凋亡基因Caspase-3、8、9、Bcl2、Bax的变化进行研究,免疫组化检测Caspase-3在小肠中的表达情况,探明热应激致小肠组织损伤的分子机制。结果:在热应激中,猪的小肠绒毛顶端上皮细胞脱落;Caspase-3、8、9、Bax基因表达上调,Bcl2基因表达下调;Caspase-3蛋白在小肠绒毛顶端表达升高。结论:热应激过程中,猪的小肠形态学损伤,细胞凋亡明显,绒毛顶端凋亡严重;死亡受体、线粒体途径被激活,促凋亡作用增强,抗凋亡作用减弱。     

金针菇多糖抑制肝癌HepG2细胞增殖并诱导细胞凋亡

MTT法检测金针菇多糖(FVP)在不同浓度(0,50,100,200,500,1 000μg/mL)及不同时间(24 h和48 h)对肝癌细胞HepG2增殖的影响;利用Annexin V-FITC/PI双染试剂盒检测FVP对肝癌细胞凋亡的影响; Hoechst33258荧光染色法检测细胞形态学变化;试剂盒法检测凋亡蛋白Caspase3、8、9活性变化。结果表明:FVP以剂效时效关系抑制细胞增殖,浓度在500,1 000μg/mL处理48 h后细胞抑制率分别为47. 2%和57. 1%。细胞凋亡率随着FVP浓度增高而增高,浓度为500μg/mL和1 000μg/mL时的凋亡率分别为33. 8%和40. 7%。FVP使细胞呈现致密、明亮、蓝色浓染的核固缩细胞凋亡变化,且镜下细胞数目随FVP浓度增加而减少。FVP能够同时使Caspase3、8、9活性蛋白表达增高,且呈浓度依赖性。FVP能抑制癌症细胞增殖并诱导细胞凋亡,其作用机制可能涉及线粒体及死亡受体途径,具体作用机制有待进一步研究。     

细胞凋亡信号传导通路的研究进展

细胞凋亡是机体维持自身稳定的一种基本生理机制,是有许多基因产物及细胞因子参与的一种有序的细胞自我消亡形式。细胞凋亡发生过程的启动和进行受到精确调控,具有独特而复杂的信号系统。各种凋亡信号通过信号传导通路传至细胞内,激活靶分子而产生细胞效应,引发细胞凋亡。一般认为,细胞凋亡存在3条主要通路:线粒体通路、内质网通路和死亡受体通路,各通路间互相联系,共同调节细胞凋亡。     

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.     

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.     

Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex

Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.     

Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion

The guanosine triphosphatase Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through interaction with effector molecules, but the molecular mechanisms by which this occurs are unknown. Here, the tethering factor p115 was shown to be a Rab1 effector that binds directly to activated Rab1. Rab1 recruited p115 to coat protein complex II (COPII) vesicles during budding from the endoplasmic reticulum, where it interacted with a select set of COPII vesicle-associated SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) to form a cis-SNARE complex that promotes targeting to the Golgi apparatus. We propose that Rab1-regulated assembly of functional effector-SNARE complexes defines a conserved molecular mechanism to coordinate recognition between subcellular compartments.     

Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor.

The signal recognition particle (SRP) directs signal sequence specific targeting of ribosomes to the rough endoplasmic reticulum. Displacement of the SRP from the signal sequence of a nascent polypeptide is a guanosine triphosphate (GTP)-dependent reaction mediated by the membrane-bound SRP receptor. A nonhydrolyzable GTP analog can replace GTP in the signal sequence displacement reaction, but the SRP then fails to dissociate from the membrane. Complexes of the SRP with its receptor containing the nonhydrolyzable analog are incompetent for subsequent rounds of protein translocation. Thus, vectorial targeting of ribosomes to the endoplasmic reticulum is controlled by a GTP hydrolysis cycle that regulates the affinity between the SRP, signal sequences, and the SRP receptor.     

ER tubules mark sites of mitochondrial division

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.     

Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor

The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.     

PML regulates apoptosis at endoplasmic reticulum by modulating calcium release

The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.