细胞凋亡在肾脏疾病中的作用

细胞凋亡是基因调控的细胞生理性死亡形式 ,是一种主动、自发性的过程 ,是目前生物学和医学界的研究热点。文章通过对细胞凋亡的形态学、生物化学、免疫化学和分子生物学的检测方法、促进细胞凋亡和抑制细胞凋亡的基因、以及细胞凋亡与肾脏疾病的关联作一综述。旨在阐明细胞凋亡在肾小球肾炎、肾小管疾病以及继发性肾脏疾病等肾脏疾病发病机制中的重要作用     

柞蚕核型多角体病毒对Tn-Hi5细胞的感染性及抗细胞凋亡分析

细胞凋亡是昆虫宿主细胞抗病毒感染及控制病毒复制增殖的一个复杂的分子生物学机制。前期研究发现柞蚕核型多角体病毒(Ap NPV)的重组病毒Ap NPV-Δph/egfp+能够感染非特异性宿主细胞Tn-Hi5,并表达EGFP蛋白。利用实时荧光定量PCR方法进一步检测分析Ap NPV-Δph/egfp+在Tn-Hi5细胞中的复制增殖特点以及子代病毒的感染性;通过检测细胞凋亡信号通路中类caspase-3蛋白酶活性,分析Ap NPV-Δph/egfp+感染Tn-Hi5细胞后对抗细胞凋亡的作用途径。结果显示,随着病毒感染时间的延长,Ap NPV-Δph/egfp+在Tn-Hi5细胞中的DNA拷贝数增加,被感染的Tn-Hi5细胞能够产生子代病毒,但病毒DNA拷贝数逐代减少;Ap NPV-Δph/egfp+感染Tn-Hi5细胞后可抑制放线菌素D诱导的细胞类caspase-3蛋白酶活性。研究结果证实Ap NPV能够在Tn-Hi5细胞中复制增殖,并具有抑制由放线菌素D诱导的细胞凋亡的作用。     

细胞凋亡的主要检测方法研究进展

细胞凋亡是生理性细胞死亡过程,凋亡在维持动物机体内环境平衡中扮演着极其重要的角色.细胞凋亡的准确检测是研究细胞凋亡相关机制,以及评价细胞凋亡在相应组织或疾病中影响的第一步.形态学是检测细胞凋亡的最直观的方法,至今一直作为细胞凋亡检测的金标准.DNA片段化检测主要有DNA ladder,TUNEL检测法和ELISA检测法...     

细胞凋亡的研究及进展

细胞凋亡(apoptosis)又称细胞程序性死亡(programmed cell death)是多细胞动物生命过程中必不可少的正常过程，它与细胞增殖具有同样重要的意义。它与生物体的生长发育以及生物体疾病的产生和控制都有极大的关联。因此，这一领域的研究受到了的生命科学研究的广泛重视，特别是近年来其研究发展非常迅速，研究的方法及检测手段都有了很大的提高，本文将从细胞凋亡的定义、形态学特征、诱导、检测方法、分子调控等方面综合分析国内外的研究进展。     

细胞凋亡常用检测方法的研究进展

近年来,细胞凋亡一直是生命科学和医学领域各专业的研究热点。作者综述了细胞凋亡的形态学、生物化学及分子生物学、酶学、免疫学和流式细胞仪法等常用的细胞凋亡检测方法,并对各种方法的优点及局限性进行了简要概述。     

山羊副流感病毒3型通过Caspase途径诱导气管上皮细胞凋亡

旨在揭示山羊副流感病毒3型(CPIV3)感染气管上皮细胞后是否可诱导细胞发生凋亡,并对细胞凋亡的信号通路进行初步探究。本研究将CPIV3病毒液接种气管上皮细胞,在12、24、36、48、72和96 h收集培养物上清检测病毒增殖滴度;通过形态学观察CPIV3诱导气管上皮细胞病变(CPE)情况;采用Annexin V-FITC/PI双染细胞凋亡检测试剂盒和Caspase-3、Caspase-8和Caspase-9活性检测试剂盒检测凋亡水平及相关指标;荧光定量PCR检测细胞凋亡分子mRNA表达水平;Western blot分析激活型Caspase-3蛋白表达变化情况。结果显示,CPIV3在气管上皮细胞中的增殖呈上升趋势,96 h能达到10~(4.50)TCID_(50)·mL~(-1);形态学观察发现,病毒接种后48 h出现细胞收缩变圆、脱落等CPE现象;流式细胞术检测及Caspase活性检测表明,感染组细胞出现细胞凋亡,48 h后细胞凋亡率达19.66%,且Caspase-3、Caspase-8和Caspase-9活性随着时间延长逐渐升高;死亡受体凋亡途径和线粒体凋亡途径细胞凋亡因子mRNA表达上调。Western blot分析揭示,激活型Caspase-3蛋白在病毒感染过程中被活化。本研究证实CPIV3感染可诱导气管上皮细胞凋亡,且Caspase途径在病毒诱导细胞凋亡的过程中发挥重要作用。     

细胞凋亡的特征及其检测方法

细胞凋亡是动物机体组织清除受损、衰老或多余细胞的一种自杀性过程,它对于保持机体各组织器官正常生长、发育、结构、功能动态平衡和维持内环境相对稳定等都具有重要意义。发生凋亡的细胞大多数是生物个体中多余或老化的细胞,有些也是发生肿瘤时的细胞或受到病毒或细菌感染的细胞。根据不同的检测方法来检测不同类型的细胞凋亡,进而来研究细胞凋亡的现象和发生机制,为揭示机体与细胞凋亡的关系和疾病与凋亡的关系提供理论依据。文章对凋亡细胞的特征、细胞凋亡各种检测方法的检测原理和结构判定,分别进行了概述。     

动物细胞大规模培养中细胞凋亡检测方法的研究

为了探讨动物细胞大规模培养中细胞凋亡的检测方法,以不同浓度氯化铵诱导BHK-21细胞凋亡,每隔12 h收集细胞,采用台盼兰形态学排染法、吖啶橙和溴化乙锭双重荧光染色法、FITC-Annexin V/PI双染色荧光显微镜观察法、原位缺口末端标记法(TUNEL)、DNA凝胶电泳、流式细胞术(FCM)法进行细胞凋亡的检测。通过比较发现,不同方法检测结果都存在着显著性差异,但吖啶橙和溴化乙锭双重荧光染色法是一种简单、易行、准确、全面定性细胞凋亡的较为可靠的方法,而FITC-Annexin V/PI双染色流式细胞仪能特异地、准确地检出早期凋亡的细胞,是较为理想的凋亡定量检测方法。这2种方法的联合运用更适应于动物细胞大规模培养过程中细胞凋亡的检测。     

细胞凋亡与疾病

早在1972年Kerr等已发现从细胞形态、超微结构和生化变化等方面来分析,细胞有二种死亡形式,一种是早被熟知的细胞坏死（Necrosis）,另一种是细胞凋亡（Apoptosis）。细胞凋亡是正常的生理过程,但是凋亡过多或过少都可引起疾病发生。因此,近年来对于细胞凋亡与疾病间关系的研究,已成为医学界的关注热点。细胞凋亡是机体维持细胞群体数量稳态的重要手段,细胞凋亡失调（凋亡不足或／和凋亡过度）可成为某些疾病的重要发病机制。     

细胞凋亡与疾病

早在1972年Kerr等已发现从细胞形态、超微结构和生化变化等方面来分析,细胞有二种死亡形式,一种是早被熟知的细胞坏死（Necrosis）,另一种是细胞凋亡（Apoptosis）。细胞凋亡是正常的生理过程,但是凋亡过多或过少都可引起疾病发生。因此,近年来对于细胞凋亡与疾病间关系的研究,已成为医学界的关注热点。细胞凋亡是机体维持细胞群体数量稳态的重要手段,细胞凋亡失调（凋亡不足或／和凋亡过度）可成为某些疾病的重要发病机制。     

细胞凋亡与疾病防治

细胞凋亡是受到包括基因在内的多种因素调节的细胞主动死亡的过程，涉及到一组半胱氨酸蛋白酶家族（caspases)对细胞凋的发生起着关键性作用，通过激活或者抑制caspases的活性，即可促进或抑制细胞发生凋亡，这就为疾病的防治提供了可能性，本文学就细胞凋亡的形态改变、发生机制作了简单综述。     

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.     

The apoptosis-necrosis continuum: insights from genetically altered mice

Apoptosis can be defined as a carefully regulated process, characterized by specific morphologic and biochemical features. It is initiated by both physiologic and pathologic stimuli, and its full expression requires a signaling cascade in which caspase activation plays a central role. Knockout mice lacking key genes encoding proteins constituting the core apoptotic cascade have helped us to establish the functional hierarchy of the mechanisms controlling apoptosis in animal development and, to a lesser extent, in disease. Induced mutant mice have also revealed the intimate crosstalk between apoptotic and other homeostatic pathways and have defined distinct temporal and tissue-specific roles of individual apoptotic effectors. Eliminating genes controlling caspase-dependent apoptosis can convert an apoptotic phenotype to a necrotic one, both in vitro and in vivo. This suggests that necrosis and apoptosis represent morphologic expressions of a shared biochemical network through both caspase-dependent mechanisms as well as non-caspase-dependent effectors such as cathepsin B and apoptosis-inducing factor. The cell death program, whether by apoptosis or necrosis, is mediated through an integrated cascade, which can be accessed at multiple sites, and propagated through numerous branch points. An understanding of the physiologic conditions that influence these decisions is required to adequately prevent, or induce, cell death.     

动物胎盘细胞凋亡的研究进展

细胞凋亡是由基因编码决定的细胞主动死亡,参与机体生理过程及疾病的发生过程。胎儿的生长发育是一个极其复杂的生理过程,在此妊娠过程中胎盘的细胞增殖和凋亡之间处于不同的平衡状态,这种状态一旦被破坏就会出现不同的病理妊娠症状。本文对影响动物胎盘细胞凋亡的因素、正常的胎盘细胞凋亡、胎盘细胞凋亡对妊娠和胎儿发育的影响进行了综述。     

Apoptotic Changes in the Epithelium Germinativum of the Cat (Felis catus s. domestica, L. 1758) at Different Ages and Breeding Seasons

Apoptosis (programmed cell death) could be considered as a physiological process that takes part in a healthy organism, which helps to maintain organism homeostasis. The visible deterioration of semen quality and the number of germ cells is accompanied by a seasonal decrease of the reproductive activity in some species. This post-effect cascade is caused by apoptosis, which is the primary mechanism responsible for the elimination of germ cells during spermatogenesis. The aim of our study was to assess apoptotic changes in the epithelium germinativum in cat testes at different ages. One hundred and two pairs of testes were obtained from domestic cats aged between 4 months and 10 years. The paraffin-embedded tissue sections were labelled using the Oncogene and Calbiochem Research Products DNA Fragmentation Detection Kit (Cat# QIA21; Darmstadt, Germany), which allows the recognition of apoptotic nuclei in tissue sections with Fragment End Labelling (FragEL^TM) of DNA. The activity of apoptotic processes in cat testes collected from the spring-summer period compared with the autumn-winter season revealed that, 59.42% and 51.51%, respectively, males testes were characterized by insignificant changes. The obtained data revealed a distinctive apoptotic changes in the young animal testes before spermatogenesis onset. An intensification of programmed death cells in the epithelium germinativum in the elder cats (between 3–6 and 6–10 years) was not observed. Apoptotic changes slightly intensified in cats aged between 12 and 36 months.     

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

病毒感染与细胞凋亡

细胞凋亡是机体在生长发育、细胞分化和病理过程中由基因编码调控的细胞主动自杀过程。机体通过凋亡将衰老、畸变和病变的细胞清除。因而 ,凋亡在生理及病理过程中具有同样重要的作用。近年来的研究发现 ,一些病毒及其编码的产物与宿主细胞的凋亡关系密切。研究病毒与细胞凋亡的相互关系 ,不仅有助于深入理解病毒的致病机理 ,开发新的抗病毒制剂 ,还为探索细胞内分子事件打开了窗口 ,而且对治疗人类病毒性疾病意义深远     

Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.