细胞色素C与细胞凋亡研究进展

在细胞凋亡过程中,线粒体是调控细胞凋亡的中心,而细胞色素C(Cyt-C)从线粒体的释放则起着关键性作用。细胞色素C释放到胞质后可激活Caspase,引发级联反应,从而导致凋亡;Bcl-2蛋白家族具有调控细胞色素C释放的功能;凋亡诱导因子AIF保证着凋亡的有序进行;内质网(ER)通过应激、招募、活化等提高线粒体对促凋亡因子的敏感性,从而使细胞色素C从线粒体线粒体膜间隙(intermembrane space,IMS)释放,发挥了重要的作用。     

线粒体功能调控机制进展及研究方法

线粒体是细胞代谢的中枢,参与细胞能量供应、新陈代谢以及凋亡、免疫等机体功能,也是细胞在各种压力下的第1道防线。维持蛋白质正确的折叠和质量控制对维持线粒体功能及细胞存活具有重要意义。因此,本文对线粒体功能损伤的调控机制（主要阐述线粒体自噬和线粒体未折叠蛋白反应）的研究进展和线粒体功能研究方法等方面进行了综述,旨在为进一步促进以线粒体为靶标的营养调控剂在动物生产上的应用提供参考。     

低氧适应的线粒体调控机制研究进展

氧气对于生物体不可或缺,当细胞面临低氧胁迫时通过诱导低氧基因做出应答。长期以来线粒体都被认为是氧浓度感受器,其呼吸链为低氧信号产生所必需,但具体机制仍不清楚,可能通过呼吸链产生ROS调控PHD活性或ROS、NO共同作用蛋白酪氨酸硝化反应产生低氧信号;线粒体基因突变和细胞代谢通路改变、呼吸链效率调控也在机体低氧适应过程中发挥重要作用。本文对线粒体如何参与生物低氧适应的机制进行综述,以期引起更多研究者对生物低氧适应的关注和对线粒体更深入系统的研究。     

寄生性原虫影响宿主上皮细胞凋亡研究进展

凋亡是宿主与寄生虫长期进化过程中形成的调节机制之一，对寄生虫感染引起宿主先天免疫反应具有重要的调控作用。隐孢子虫、十二指肠贾第虫和芽囊原虫等寄生性原虫感染可诱导宿主胃肠道上皮细胞发生凋亡。NF-κB信号通路和非编码RNA等参与调控隐孢子虫感染诱导的宿主上皮细胞凋亡，已报道的凋亡途径包括Fas/FasL和TRAI/TRAIL途径；十二指肠贾第虫感染通过内、外两种凋亡途径调控宿主上皮细胞的凋亡，TNFR1信号通路参与细胞凋亡的调控；芽囊原虫和十二指肠贾第虫感染在诱导宿主上皮细胞凋亡水平上存在虫株间的差异。因此，探索隐孢子虫、十二指肠贾第虫和芽囊原虫诱导宿主上皮细胞凋亡机制，对研究寄生性原虫入侵机制及开发设计抗寄生性原虫药物和疫苗具有重要意义。     

线粒体与细胞凋亡的关系

线粒体是细胞能量合成和贮存及物质代谢、能量转化的重要场所,不仅能诱导细胞凋亡,还是细胞凋亡的执行者。在细胞凋亡过程中,与凋亡相关的活性物质,如细胞色素C（Cyt·c）、凋亡诱导因子（AIF）、B细胞淋巴瘤基因-2（Bcl-2）等从线粒体释放到膜间隙中,使线粒体膜通透性转换孔（mitochondrion permeability transition pore,MPTP）开放,削弱线粒体膜两侧的质子梯度,导致线粒体膜电位降低,诱导细胞凋亡。同时线粒体内的Bcl-2家族对细胞凋亡具有调节作用。笔者就MPTP、Cyt·c、AIF、Bcl-2与细胞凋亡的关系进行了综述。     

受体相互作用蛋白1(RIP1)对BCG诱导小鼠巨噬细胞凋亡的调控作用

旨在通过构建受体相互作用蛋白1(RIP1)腺病毒干扰载体,研究其对BCG诱导的RAW264.7细胞凋亡相关指标的影响,以探讨其在BCG诱导RAW264.7凋亡过程中的调控作用。笔者构建RIP1腺病毒干扰载体,并转染感染BCG的小鼠RAW264.7细胞系,利用流式细胞仪检测各处理细胞凋亡率、细胞线粒体膜电位、细胞活性氧水平及细胞周期等指标,并用Western blot检测凋亡相关蛋白的表达水平。结果显示:BCG感染显著上调了RIP1的蛋白表达水平并提高了小鼠巨噬细胞RAW264.7的凋亡率,当RIP1被干扰后,BCG感染后的RAW264.7细胞凋亡率和活性氧水平显著降低,而促凋亡蛋白Bax表达量显著下调,线粒体膜电位和抑凋亡蛋白表达量上调。同时,BCG感染后细胞周期滞留于G_1期。BCG感染可有效上调RIP1表达量并诱导RAW264.7细胞凋亡。RIP1通过下调BCG感染后RAW264.7细胞的线粒体膜电位,上调活性氧含量并提高凋亡相关蛋白Bax/Bcl-2比值,使细胞周期阻滞于G_1期从而参与诱导细胞凋亡。     

VDAC1的生物学功能及其在病毒感染中的作用机制研究进展

电压依赖性阴离子通道1(VDAC1)是线粒体外膜上含量极为丰富的一种孔道蛋白，是线粒体功能的重要调节剂，除了协调代谢产物、胆固醇和脂肪酸等在线粒体膜上转运外，还参与细胞凋亡、线粒体DNA释放和氧化应激调控等过程。在病毒感染细胞后，VDAC1的表达或寡聚化水平发生变化，进而调控细胞与病毒的相互作用，故被认为是抗病毒治疗的潜在药物靶点。本文就VDAC1的生物学结构、功能及其在病毒感染中的作用机制进行综述，以期为进一步研究VDAC1参与病毒感染机制的研究提供理论参考。     

Fas对镉激活PC12细胞线粒体凋亡通路的影响

旨在探究死亡受体Fas在镉致大鼠肾上腺嗜铬细胞瘤细胞（PC12）凋亡中的作用及其对线粒体通路的调控机制,用10 μmol·L^-1镉处理Fas基因沉默的PC12细胞株12 h,通过Western blot检测BH3相互作用域死亡激动剂（BID）、半胱氨酸蛋白酶-9（caspase-9）、半胱氨酸蛋白酶-3（caspase-3）、多聚二磷酸腺苷核糖聚合酶（PARP）的活化情况,Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤/白血病-2基因（Bcl-2）、凋亡诱导因子（AIF）、核酸内切酶G（Endo G）的表达情况,以及细胞色素C（Cyt C）在细胞内的分布情况,免疫荧光染色检测AIF核转位。结果显示,镉极显著上调tBID/BID比值和Bax/Bcl-2比值,诱导Cyt C从线粒体释放到细胞质,激活caspase-9、caspase-3和PARP,增加AIF和Endo G蛋白表达水平（P<0.01）,并诱导AIF核转位;沉默Fas极显著抑制镉引起的tBID/BID比值和Bax/Bcl-2比值升高,Cyt C从线粒体释放到胞浆,caspase-3、PARP蛋白活化和AIF、Endo G蛋白表达水平极显著升高（P<0.01）,显著抑制镉激活的caspase-9（P<0.05）,并抑制AIF核转位。综上表明,Fas通过调控线粒体通路参与镉致PC12细胞凋亡。     

线粒体膜通透性变化与细胞凋亡的关系

线粒体膜通透性转换孔是一种位于线粒体内膜的非选择性孔道,它的开放引起线粒体膜通透性改变,导致细胞色素C、凋亡诱导因子和Ca²⁺及膜间隙中的胱冬肽酶原等凋亡因子释放到细胞质中,致使细胞整体结构破坏、功能紊乱,发生凋亡。以前普遍认为线粒体膜通透性改变(mitochondrial permeability transition,MPT）是导致细胞凋亡的关键点,但一些新的研究结果表明,MPT与细胞凋亡并不存在必然的联系。作者结合细胞凋亡方面的研究着重就线粒体膜通透性转换的生物学功能和腺嘌呤核苷酸转位子、环孢素A 结合蛋白D及Bcl-2家族蛋白定位、转位在通透性转换中的作用及其与细胞凋亡的关系进行概括性论述与分析。     

线粒体与细胞凋亡

细胞凋亡是一种由基因控制的细胞自杀性死亡过程,是机体维持自身稳定的一种基本生理机制。机体通过细胞凋亡清除损伤、衰老与突变的细胞,维持生理平衡。通过对细胞凋亡的研究发现,线粒体在调节细胞凋亡中发挥着关键作用。尤其是被称为细胞凋亡主开关的线粒体通透性改变孔(MitochondrionPermeabilitytransitionPore,MPTP),它是细胞色素C、Smac、AIF等凋亡诱导因子的主要来源。本文以MPTP的开放、细胞色素C、AIF在细胞凋亡中的作用以及Bcl-2家族对细胞凋亡的调控做一综述,以便广大读者能系统地了解线粒体在细胞凋亡中的作用。     

Sensitivity of skeletal muscle to pro-apoptotic factors

In mononuclear cells, apoptosis leads to DNA fragmentation and cell destruction, regardless of the activated pathway. As regards multinuclear cells, e.g. skeletal muscle fibers, apoptosis rarely induces the death of the entire cell, and it generally affects single nuclei. This process, referred to as nuclear apoptosis, has a negative effect on the expression of genes in the myonuclear domain. Apoptosis may be initiated in muscle cells by external stimuli which activate cell membrane death receptors as well as by internal stimuli which stimulate the mitochondrial release of pro-apoptotic proteins. Reactive oxygen species also play an important role in the initiation of apoptosis. In muscle cells, ROS are produced in response to extracellular reactions or by cell mitochondria. It is, therefore, believed that mitochondria play a central role in apoptosis within skeletal muscle. Skeletal muscles have a well-developed system that protects them against oxidative damage. Myogenic stem cells are an integral part of multinucleated myofibers, and they are critically important for the maintenance of normal muscle mass, muscle growth, regeneration and hypertrophy. The latest research results indicate that myogenic cells are more sensitive to oxidative stress and pro-apoptotic factors than well-differentiated cells, such as myotubes. The complex structure and activity of skeletal muscle prompted research into the role of apoptosis and its intensity under various physiological and pathological conditions. This review summarizes the results of research investigating control mechanisms and the apoptosis process in skeletal muscle fibers, and indicates unresearched areas where further work is required.     

Caspases与细胞凋亡

Caspases是一类蛋白裂解酶,在细胞内以无活性的酶原形式存在。Caspases具有半胱氨酸激活位点和底物裂解位点,当作用于胞内特异性底物后将引起细胞凋亡。Caspases的底物特异性由其裂解位点N-端的4个氨基酸残基决定,其底物裂解部位通常位于靶蛋白质一级结构中Asp残基后,数目由一个到多个不等。Caspase酶系作为细胞凋亡的中枢效应器,在细胞凋亡的启动及进程中发挥着极为重要的作用。无活性的caspases酶原主要通过上游caspseses加工机制、邻近诱导机制或偶联调节亚基机制三种方式被激活而引发细胞凋亡。细胞凋亡是细胞生命活动的基本特征之一。本文就引起凋亡的酶系和细胞凋亡的生化机理加以综述。     

Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei,mitochondria, and plasma membranes in mice

Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.     

Morphological and biochemical aspects of apoptosis,oncosis and necrosis

Recent investigations have demonstrated the need for a precise differentiation of various forms of cell death such as apoptosis, oncosis, necrosis and programmed cell death. Apoptosis is marked by cellular shrinking, condensation and margination of the chromatin and ruffling of the plasma membrane with eventually breaking up of the cell in apoptotic bodies. Cell death marked by cellular swelling should be called oncosis, whereas the term necrosis refers to the morphological alterations appearing after cell death. Apoptosis and oncosis are therefore pre-mortal processes, while necrosis is a post-mortal condition. The term programmed cell death refers to the 'fixed' pathway followed by dying cells, whether or not with the characteristic morphology of apoptosis. Three mechanisms are actually known to be involved in the apoptotic process: a receptor-ligand mediated mechanism, a mitochondrial pathway and a mechanism in which the endoplasmic reticulum plays a central role. All three mechanisms activate caspases which are responsible for the characteristic morphological changes observed during apoptosis. A review of the different methods used for detecting apoptotic cells demonstrates that most of these techniques are not entirely specific.     

家禽免疫器官内细胞凋亡及其凋亡相关基因调控研究进展

细胞凋亡是生物界广泛存在的一种重要的生命过程,是多细胞动物为调节机体发育、维护内环境稳定、有基因调控的细胞主动性死亡过程。在细胞凋亡的分子生物学研究过程中,发现已有多种基因参与细胞凋亡的调控,其中包括Bcl-2家族和Bax、p53、Fas和FasL等。家禽免疫器官胸腺、脾脏和法氏囊内存在着自然凋亡现象,许多学者用物理性、化学性和生物性因素诱导了家禽免疫器官细胞凋亡,证实了细胞凋亡相关基因参与了家禽免疫器官内细胞凋亡的调控。     

No caspase activation but overexpression of Bcl-2 in bovine cells infected with noncytopathic bovine virus diarrhoea virus

Cytopathic bovine viral diarrhoea viruses (cp BVDV) induce apoptosis in permissible cell cultures via the intrinsic pathway, which involves the mitochondria as key organelles. An important event is the irreversible opening of the permeability transition pore (PTP) and the breakdown of the transmembrane potential DeltaPsi(m). The resulting release of cytochrome C from the mitochondria serves as a trigger to form the apoptosome which then leads to caspase activation and cell death. In contrast, noncytopathic (ncp) BVDV do not seem to affect cells in vivo or in vitro, suggesting that they inhibit apoptosis. Interestingly, inhibition of caspases in cells infected with cp BVDV delayed the apoptotic cascade but did not prevent the cytopathic effect (CPE). This suggests that the induction of apoptosis and the processes finally leading to the CPE may proceed separately, implying that the inhibition of apoptosis by ncp BVDV has to start earlier in the cascade. In this study we show that in fact apoptosis inhibition in cells infected with ncp BVDV must occur at the mitochondrial level, before the activation of the caspase cascade occurs. To elucidate the role of mitochondria after infection of cells with ncp BVDV, expression of Bcl-2 and Bax were analysed. It was shown that while Bax expression was not affected, the anti-apoptotic Bcl-2 protein was upregulated, presumably suppressing initiation of cell death and enabling persistent infection in vitro.     

哺乳动物附植前胚胎细胞凋亡的研究进展

细胞凋亡是细胞为维持内环境稳定,更好地适应生存环境而主动形成的一种自我死亡过程,它涉及一系列基因的激活、表达及调控等作用。附植前的胚胎就已经观察到凋亡的发生,研究认为超出一程度的凋亡不利于胚胎的发育。作者主要对哺乳动物附植前胚胎发生凋亡的研究进行阐述,为改进体外培养体系、提高胚胎质量提供一定的理论依据。     

线粒体凋亡途径在锰致鸡支持-生精细胞凋亡中的作用

探讨线粒体凋亡途径在锰致鸡支持-生精细胞凋亡中的作用。取对数生长期鸡支持-生精细胞,用含0、2、3、4mmol.L-1MnCl2的DMEM培养液,培养24h,采用吖啶橙-溴化乙锭(AO/EB)双荧光染色法检测鸡支持-生精细胞凋亡,流式细胞仪检测线粒体膜电位(△ψm),Westernblot法检测胞浆内细胞色素C(Cytc)蛋白表达含量,分光光度法检测半胱氨酸天冬氨酸蛋白酶9,3(Caspase-9,3)活性。用含MnCl2的DMEM培养液培养24h后,处理组细胞与对照组相比,鸡支持-生精细胞凋亡指数逐渐升高(P0.01);线粒体膜电位显著降低(P0.01);胞浆中Cytc蛋白表达量逐渐增加;Caspase-9,3活性逐渐升高(P0.01)。线粒体途径介导的凋亡是锰对鸡生殖毒性作用的主要机制之一。     

凋亡素诱导的肿瘤细胞凋亡

细胞凋亡是机体维持自身稳定的一种基本生理机制 ,并贯穿于整个生命活动。细胞凋亡异常可导致一些疾病的发生 ,如肿瘤的发生。同时 ,细胞凋亡的可诱导性也为肿瘤治疗提供了新的思路。凋亡素是来源于鸡贫血病毒的一个小分子蛋白。它能够选择性地诱导肿瘤细胞的凋亡 ,而对正常二倍体的细胞无任何毒副作用 ,并且正常细胞对凋亡素具有耐受性。凋亡素诱导肿瘤细胞凋亡既不需要依赖 p53 ,也不会被bcl-2的过表达所抑制。凋亡素还能诱导人成骨肉瘤细胞多药耐药株 R-OS-73 2的细胞凋亡。这些特点使凋亡素成为一种新型的候选抗肿瘤制剂。