彗星电泳法在植物原生质体凋亡检测中的应用

应用中性法彗星电泳检测烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）原生质体的凋亡。结果表明,彗星发生的百分率（彗星率）和发生核质固缩及“核着边”（凋亡形态学标志）的细胞的百分率之间存在明确的相关性。利用标准的细胞凋亡检测手段,包括ＤＮＡｌａｄｄｅｒｉｎｇ及核糖核酸末端转移酶介导的ｂｉｏｔｉｎｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）,都证明这种彗星电泳法可以用来比较精确地检测植物原生质体的凋亡。     

细胞色素c能诱导植物细胞编程性死亡

以悬浮培养的胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）与烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．ＢＹ２）细胞原生质体为材料,加入一定浓度的细胞色素ｃ和ｄＡＴＰ。不同取样时间的ＤＡＰＩ荧光染色与电镜超薄切片观察的结果显示染色质发生凝集、趋边化,最终形成凋亡小体。核酸电泳显示ＤＮＡ发生特异降解并形成电泳“阶梯”（ＤＮＡｌａｄｄｅｒ）。用末端脱氧核糖核酸转移酶介导的ｄＵＴＰ切口末端标记方法（ＴＵＮＥＬ）检测发现ＤＮＡ的３＇ＯＨ断端被原位特异标记。以上结果说明：细胞色素ｃ能诱导植物细胞发生典型的凋亡。     

用TUNEL法检测植物原生质体的凋亡

TUNEL是近年来发展的一种对凋亡细胞进行原位检测的方法,可以特异性地标记完整的凋亡细胞核或凋亡小体的染色体3'_OH断裂末端,但在植物细胞中的应用还不多。本文报道应用TUNEL法检测胡萝卜原生质体的凋亡,并与DNA电泳、彗星电泳等方法进行了比较,结果表明它是一种适用于植物原生质体凋亡检测的灵敏度较高的方法。     

细胞凋亡原位检测的TUNEL法

细胞凋亡原位检测的ＴＵＮＥＬ法刘勇（江西省人民医院病理科，南昌３３０００６）细胞凋亡（ａｐｏｐｔｏｓｉｓ），又称程序性细胞死亡（ｐｒｏｇｒａｍｍｅｄｃｅｌｄｅａｔｈ，ＰＣＤ），是细胞的一种生理性死亡过程，有关细胞凋亡的研究近年来受到高度重视。末端转移...     

酶电泳资料和系统与进化植物学研究综述

酶电泳资料和系统与进化植物学研究综述葛颂（中国科学院植物研究所系统与进化植物学开放研究实验室北京１０００９３）关键词同工酶,电泳,植物系统学,进化ＥＬＥＣＴＲＯＰＨＯＲＥＴＩＣＤＡＴＡＡＮＤＳＴＵＤＩＥＳＯＦＰＬＡＮＴＳＹＳＴＥＭＡＴＩＣＳＡＮＤＥＶ...     

稀土元素镧在植物细胞生理研究中应用的进展

稀土元素镧在植物细胞生理研究中应用的进展刘敏,周世恭（中国科学院植物研究所,北京１０００４４）ＡＤＶＡＮＣＥＳＩＮＴＨＥＡＰＰＬＩＣＡＴＩＯＮＯＦＲＡＲＥＥＡＲＴＨＥＬＥＭＥＮＴ──ＬＡＮＴＨＡＮＵＭＴＯＰＬＡＮＴＣＥＬＬＰＨＹＳＩＯＬＯＧＩＣＡＬＲ...     

植物原生质体培养及有关生理基础问题

植物原生质体培养及有关生理基础问题赵颖,梁海曼（杭州大学生物系,杭州３１００１２）ＰＬＡＮＴＰＲＯＴＯＰＬＡＳＴＣＵＬＴＵＲＥＡＮＤＳＯＭＥＢＡＳＩＣＰＨＹＳＩＯＬＯＧＩＣＡＬＰＲＯＢＬＥＭＳ￥ＺｈａｏＹｉｎｇａｎｄＬｉａｎｇＨａｉｍａｎ（Ｄｅｐａｒ...     

植物细胞凋亡的ELISA检测(英文)

本文利用ＴＵＮＥＬ方法在单个细胞水平上对苯胺灵诱导的玉米根尖细胞死亡进行了检测。结果表明,一定浓度的苯胺灵能诱导玉米根尖细胞发生主动性的细胞凋亡,具有细胞凋亡典型的形态和生化特征即细胞核浓缩并形成核碎片、染色质边缘化及ＤＮＡ特异片段化等（Ｆｉｇ．１）。首次利用ＥＬＩＳＡ方法在群体水平上对这种细胞凋亡进行了进一步检测。结果表明：动物细胞研究常用的ＥＬＩＳＡ方法同样也适合用于植物细胞凋亡的检测。在凋亡前期,随着凋亡过程的进行,细胞质中的ＯＤ值逐渐上井（Ｆｉｇ．２）。剂量实验表明,０．２ｍｇ／ｍＬ苯胺灵最适合于诱导细胞凋亡（Ｆｉｇ．３）。     

高活性细胞激动素TDZ在植物组织培养中的应用

高活性细胞激动素ＴＤＺ在植物组织培养中的应用王关林方宏筠那杰（辽宁师范大学生物系,大连,１１６０２２）ＡＰＰＬＩＣＡＴＩＯＮＯＦＰＯＴＥＮＴＣＹＴＯＫＩＮＩＮ－ＴＨＩＤＩＡＺＵＲＯＮ（ＴＤＺ）ＴＯＰＬＡＮＴＴＩＳＳＵＥＣＵＬＴＵＲＥＷａｎｇＧｕａｎ－...     

青春双歧杆菌DM8504菌株对小鼠体内HCa-F_(25)/16A_3-F肿瘤细胞抑制作用的初步研究

本实验应用加热处死的青春双歧杆菌ＤＭ８５０４菌株皮下注射荷瘤ＨＣａ－Ｆ２５／１６Ａ３－Ｆ肿瘤的ＢＡＬＢ／ｃ小鼠,酶联法测定小鼠体内ＴＮＦ－α、ＩＬ－６的含量,ＴＵＮＥＬ法及电镜观察肿瘤组织中是否有凋亡细胞的存在。实验结果指出：双歧杆菌能提高荷瘤小鼠体内ＴＮＦ－２的含量,较对照组明显升高,但ＩＬ－６的含量处理组与对照组之间无显著性差异。ＴＵＮＥＬ法除观察到不同程度的坏死组织外,未见到散在的凋亡的肿瘤细胞,电镜观察与ＴＵＮＥＬ结果相一致,肿瘤细胞呈现坏死细胞的超微结构,未见凋亡细胞所具有的典型的形态特征。结果提示：双歧杆菌通过调节机体的免疫系统发挥抗肿瘤的作用,其对ＨＣａ－Ｆ２５／１６Ａ３－Ｆ肿瘤细胞的杀伤是通过坏死的方式实现的。     

Application of Comet Assay in Plant Protoplast Apoptosis Detection

The authors report the application of neutral comet assay in the detection of apoptosis in tobacco (Nicotiana tabacum L. ) pretoplasts. The results suggested a close inter-relationship between comet formation and nuclear compacting into densed masses at the nuclear periphery (a typical morphological symptom of apoptosis). Standard detection of hallmarks of apoptosis, including DNA laddering and TdT-mediated biotin-dUTP nick end-Lase Labeling (TUNEL), was also performed in order to conform the reliability of comet assay in the detection of apoptosis in plant protoplasts.     

Possible involvement of the mitochondrial alternative pathway in ethylene-induced apoptosis in tomato protoplasts

In this study, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to detect the expression of alternative oxidase (AOX) protein in tomato mitochondria. The results show that there was an obvious correlation between the ethylene-induced apoptosis and the levels of AOX protein in tomato cells undergoing ethylene-induced apoptosis. In addition, when tomato protoplasts were preincubated with 2 mM salicylhydroxamic acid (SHAM), an inhibitor of the alternative pathway, before their exposure to ethylene, the TUNEL positive reaction and DNA fragmentation were obviously accelerated. We suggest that AOX may play an important role in protecting tomato protoplasts against ethylene-induced apoptosis.     

Method of specific detection of apoptosis using formamide-induced DNA denaturation assay.

We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide-MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 3'-OH DNA ends, whereas the formamide-MAb assay detects ssDNA itself (25-30 bp). We found that the formamide-MAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 3'-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 3'-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/nuclear area) obtained by the formamide-MAb assay. On the basis of these findings, we conclude that the formamide-MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide-MAb assay was slightly weaker than that of the TUNEL method.     

羟自由基诱导的烟草原生质体的凋亡:流式细胞法的新证据

用1.0 mmol/L FeSO4/0.5 mmol/L H202处理烟草(Nicotiana tabacum L.cultivar BY 2)原生质体,发现羟自由基能够诱导烟草原生质体的凋亡.具体表现为细胞核皱缩、DNA Ladder、TUNEL阳性反应等典型的凋亡特征.在动物细胞凋亡过程中,线粒体起着非常重要的作用,其中膜电位(△ψm)的变化以及由其引起的位于线粒体膜上的通透性孔(PTP)的开放与Cyt c的释放有关.另外,在动物凋亡细胞中,磷脂酰丝氨酸(phosphatidyl serine,PS)会从细胞膜内侧向外翻转.为了判断植物细胞凋亡过程中膜电位的变化情况以及PS的外翻程度,我们采用了流式细胞法.结果表明,随着处理时间的延长,烟草原生质体线粒体的膜电位逐渐降低;膜内PS大量外翻.说明由羟自由基和烟草原生质体组成的凋亡体系是一种可靠的凋亡组合,可以用来对植物细胞凋亡机理做进一步研究.     

Apoptosis detected in hybrids between Nicotiana glutinosa and N. repanda expressing lethality

Hybrid lethality is one of the mechanisms for reproductive isolation. Apoptotic features were detected in the cells of hybrid seedlings of Nicotiana glutinosa L. ×N. repanda Willd. Condensation of chromatin and fragmentation of nuclei were observed in the leaf protoplasts isolated from hybrid seedlings expressing this lethality. Fragmentation of nuclei was correlated with the progression of lethal symptoms, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid leaves showing lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation associated with nuclear fragmentation. In-situ detection of DNA fragmentation using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in all leaf cells. This is the first evidence that apoptosis can induce suicide of hybrid plants, thus leading to reproductive isolation. Received: 18 June 1999 / Accepted: 20 July 1999     

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.     

Effect of ethrel on apoptosis in carrot protoplasts

In recent years, apoptosis has been reported to exist in plants during normal development and in response to stress. However, little is known about the relation of hormones to this form of programmed cell death. Here, we report examination of characteristics of apoptosis in carrot protoplasts induced by ethylene evolved from ethrel (2-chloroethylphosphonic acid). Nucleus condensation and DNA ladders were observed, and neutral comet assay, which detects DNA cleavage, also provided evidence that ethrel treatment resulted in nuclear DNA fragmentation. Strikingly, a close correlation between the incidence of DNA comets and the percentage of apoptotic protoplasts was shown in ethrel-treated carrot protoplasts. In conclusion, this study demonstrated that ethylene is an active inducer of apoptosis in carrot protoplasts, and that ethylene-induced plant cell death showed characteristics similar to those of apoptosis in animals.