赵 允

论文题目：植物非细胞体系的建立及凋亡机理的研究

作者简介：赵 允，男，1971年01月出生，1996年09月师从于北京大学翟中和教授，于2001年06月获博士学位。

摘 要

I. 植物非细胞体系的建立及凋亡机理的研究

与动物细胞相同，植物细胞的细胞编程性死亡也是一种由细胞内部基因控制的、普遍存在的主动性死亡行为，在植物的系统发育、组织分化、环境压力以及病原体感染过程中起到了重要的作用。动物细胞中，从人类到线虫，再到昆虫，凋亡都是一个相当保守的并由基因调节的生命过程。但迄今为止，植物细胞中是否存在一个类似的高度保守的凋亡信号通路来引发各种凋亡现象仍不清楚。这很大程度上是因为没有建立良好的、适于植物细胞凋亡机理研究的体外实验模式。由于利用体内细胞研究凋亡非常困难，细胞同步性低，凋亡时间短，难于操作与检测。非细胞体系作为一种研究凋亡的有效的辅助手段可以很好的解决这一问题。在本实验室多年进行动物非细胞体系核重建研究的基础上，我们建立了基于胡萝卜悬浮培养细胞的植物非细胞体系，在该体系中加入爪蟾卵的去膜精子及膜泡组分，可以成功地诱导细胞核的体外重建。形态学的证据表明，重建的细胞核具有典型的正常真核细胞核的特征，例如双层核膜及核孔复合体。同时，利用小球菌核糖核酸酶处理的结果表明，去膜精子在此非细胞体系中发生了结构改建并且在重建过程中形成了核小体结构。利用植物的非细胞体系进行核重建尚未见相关报道，我们的实验结果初步证明细胞核的自组装现象在植物非细胞体系中也能实现，说明细胞核的自组装在动植物系统中均是一个广泛存在的现象，同时也为进一步应用非细胞体系进行植物细胞凋亡机理的研究打下了一个良好的实验基础。

在胡萝卜细胞胞浆提取物CS-100中加入细胞色素C可成功诱导小鼠肝细胞核出现染色质凝集、趋边化、产生类似凋亡小体的结构等明显的凋亡特征。同时，利用TUNEL方法可特异标记凋亡核的3'-OH断裂末端。经诱导后DNA发生了特异性降解，形成了典型的DNA ladder。统计学的数据表明，在该体系中，细胞色素C可以高效的诱导胡萝卜细胞核的同步化凋亡,表明CS-100非细胞体系是研究植物细胞凋亡机理的一个很好的、方便的实验模式。另一方面，以DEVD-pNA检测Caspase活性的结果也表明在植物非细胞体系中存在Caspase-3-like活性。Caspases的特异抑制剂Ac-DEVD-CHO及Ac-YVAD-CHO可以特异性的抑制DNA Ladder的产生。同时，在凋亡过程中核纤层蛋白特异的由88kDa和66kDa降解成47KD和37KD两个片段。这些结果证明细胞色素C在动植物细胞凋亡过程中均起到了重要的作用，在植物细胞中存在与动物细胞相似但不完全相同的凋亡信号转导途径。

Caspase-3作为凋亡效应分子，在动物细胞凋亡进程中处于中心地位。同时，Caspases家族蛋白酶具有一旦在细胞中过表达既可自身激活并诱导细胞凋亡的特性。然而，我们在植物细胞凋亡的过程中始终不知道是否在植物细胞中存在Caspase的同功酶；并且，如果存在，他们在植物细胞凋亡过程中扮演何种角色。在我们的实验结果显示在植物系统内可能同样存在Caspase-3的同功酶。为了进一步研究Caspase-3在植物系统内细胞凋亡过程中所扮演的角色，我们将人源的caspase-3基因通过抽真空的方法在土壤农杆菌的介导下转入至烟草叶片中，结果显示人源caspase-3基因在烟草细胞中的过表达可以诱导烟草叶片发生细胞编程性死亡。当caspase-3基因在烟草叶片中过表达，可以在叶片表面诱导产生由死亡细胞所形成的枯斑。形态学的证据显示了典型的细胞凋亡特征；TUNEL实验及电泳结果显示了凋亡过程所特有的3’-OH断裂及DNA Ladder的形成。另一方面，PARP蛋白作为一种重要的核蛋白及活化Caspase-3的直接底物也同时被降解。所有的这些证据不仅说明动物caspase-3基因的表达可以诱导烟草细胞的编程性死亡，也进一步说明在植物细胞中存在Caspse-3的同功酶以及相关信号转导通路。

细胞核在凋亡过程中的形态学变化是凋亡的一个重要事件，具有典型的特征。但由于对凋亡的形态学观察主要都是在整体细胞水平进行，因此人们对凋亡过程中细胞核的出泡现象及其原因了解甚少。我们利用非细胞体系去除细胞膜及细胞器的独特特点对凋亡过程中细胞核的变化，如染色质凝集、边缘化、凋亡小体及DNA Ladder的产生，进行了精细的观察，以期解释凋亡过程中的细胞核出泡现象的原因及其分子基础。透射电子显微镜及扫描电子显微镜的结果说明在凋亡小体产生之前有不含染色质的较小的空泡产生，而凋亡小体的产生是一个相对较晚的事件。当在凋亡诱导体系内加入Caspase-6的特异抑致剂AC-YVAD-CHO，这种空泡及凋亡小体不能够产生。我们进而使用整装电镜的技术对实验结果进行了观察，发现尽管细胞核内的核纤层在凋亡的早期发生了降解，但核基质在凋亡过程中大部分保持完整。这些结果表明核纤层在凋亡小体结构的保持及凋亡小体与凋亡细胞核的连接上均起到了重要的作用。

II. 爪蟾卵非细胞体系中磷酸肌酸对细胞凋亡的影响

细胞凋亡过程中，Caspases家族蛋白酶活化及其底物的降解导致了一系列典型的凋亡形态学变化及生化反应。凋亡细胞线粒体中的细胞色素C释放至胞浆中，与Apaf-1一起在dATP或ATP的辅助下激活Caspase-9。活化的Caspase-9对procaspase-3进行剪切并导致其活化。活化的Caspase-3可以对其底物进行特异性降解。在这些Caspase-3凋亡底物中procaspase-6与DFF45尤为重要。Procaspase-6受到Caspase-3的剪切后活化，并切割细胞核内的Lamin蛋白，导致细胞核的一系列形态学变化；而DFF45降解释放出DFF40，将染色质DNA切割，电泳形成DNA Ladder。

我们实验室利用爪蟾卵非细胞体系建立了成熟的凋亡诱导体系。在爪蟾卵提取物中，加入细胞色素C及小鼠肝细胞核可以高效的诱导这一系列凋亡反应的发生。由于爪蟾卵非细胞体系是一个成熟的细胞周期研究体系，在加入ATP再生系统后（ATP、磷酸肌酸、磷酸肌酸激酶），可以诱导各种不同细胞周期时相的发生。我们于是在这种非细胞体系研究了磷酸肌酸对细胞核凋亡的影响，发现当磷酸肌酸加入到这种凋亡诱导体系中，可以加速凋亡的形态学变化，但却抑制了DNA Ladder的产生。我们检测了该凋亡体系中Caspase-3的活性，结果表明，磷酸肌酸的加入增强了Caspase-3的活力。这种活力的增加可能是由于系统内ATP水平的增加所导致。我们继而分析核纤层蛋白的变化，Western Blotting结果显示Lamin A、C蛋白在磷酸肌酸加入后降解加剧，这可能是导致形态学变化加剧的原因。由于DNA Ladder的产生与DFF45的降解以及DFF40的释放直接引起，我们已经证实了在Xenopus egg提取物存在DFF45-like蛋白及凋亡相关的特异DNase，凋亡过程中，DFF45被降解，释放出活化的DNase切割染色质DNA形成DNA Ladder，所以我们进一步检测系统内的DFF45-like蛋白的变化，发现在磷酸肌酸加入后，该蛋白的降解受到了抑制。表明DNA Ladder的抑制是由DFF45-like蛋白没有被降解所致。为了检测磷酸肌酸对凋亡的影响是否是系统内ATP水平的增加及pH的降低所造成，我们在系统内加入了外源的ATP及多种有机酸。结果显示ATP水平的增加及有机酸的加入并不能导致对DNA Ladder形成的抑制。另一方面，磷酸肌酸对DNA Ladder形成的抑制具有时间依赖性，说明系统中某种或某些因子由于磷酸肌酸的加入而受到了影响并导致DNA Ladder形成的抑制。考虑到磷酸肌酸可以提供磷酸根，我们将磷酸化抑致剂LiCl加入到体系中，发现细胞核的形态学变化减缓至正常速度，DFF45-like蛋白被降解，染色质DNA重新被降解形成DNA Ladder。表明磷酸肌酸的加入可能导致了系统内部蛋白发生磷酸化，其中包括DFF45-like蛋白，使其不能被Caspase-3切割，最终造成了DNA Ladder形成的抑制。

关键词：非细胞体系，核重建，细胞凋亡，Lamin，Caspase3，磷酸肌酸， DFF45

Abstract

I. The construction of plant cell-free system and research in the mechanism of apoptosis

Just as in animal cells, the programmed cell death of plant cells is a kind of gene-controlled, ubiquitous and active cell death. It also plays important roles during normal development, tissue differentiation, environmental stress, and pathogen attack. The concept of a functionally conserved, gene-directed program, PCD, also termed as apoptosis, for the maintenance of cellular homeostasis that is dependent on localized cell death is established in animal systems, including humans, nematodes, and insects. But it is still obscure whether there is(are) conserved signal transduction pathways in plants leading to cell death by specifically ordered metabolic changes. This is mainly due to the shortage of a good apoptosis model to suit the mechanism research in plant cell. For the reasons that it’s difficult to study apoptosis in vivo, so we consider using cell-free system. Based on the years of experience about the in vitro nuclear reassembly, our laboratory construct a plant cell-free system from suspended-culture of carrot cells. And we had developed this system to reassembly nuclear structure around the added demembranated sperm chromatin of Xenopus. Morphological evidences suggest that reassembled nuclei display the typical characteristics of normal eukaryotic nucleus, such as double-layered nuclear membrane and nuclear pores. Micrococcal nuclease treatment indicates that the remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution. These data indicate that the nuclear reconstitution can be induced in cell-free system from plant, and the self-assembly of nucleus is ubiquitous in both animal and plant cells. Because in vitro nuclear reassembly is an important standard to test the quality of a cell-free system, so the cell-free system from plant cell could meet further use to study mechanism of plant cell apoptosis.

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling procedure (TUNEL) was performed to detect the breakage of 3’-OH ends of a DNA strand. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. On the other hand, the result of DEVD-pNA suggested that the activation of Caspase-6-like occurred in this cell-free apoptosis inducing system. Furthermore, we found that nuclear lamins were degraded from 88kDa and 66kDa to 37kDa and 47kDa fragments. The DNA fragmentation could be inhibited by Ac-DEVD-CHO and Ac-YVAD-CHO. All these result and those results, which the carrot nuclei could be induced to undergo typical apoptosis by cytochrome c in CS-100, indicate that cytochrome c does play a important role during apoptosis in both animal and plant cells, and the apoptosis in plant cells may share similar pathways to apoptosis in animal cells.

Caspase-3 (CPP32, cysteine protease P32), as the effector, plays a central role during the apoptotic process of animal cells. When overexpressed in cells, caspases can be activated by themselves and induce apoptosis. However, we still don’t know whether their isoenzymes exist in plant cells and if so, what role they play during programmed cell death of plant cells. We herein report that the overexpression of human caspase-3 gene could induce tobacco leaf cells to undergo programmed cell death. When caspase-3 gene was overexpressed, the zone of cell death appeared on the surface of infected tobacco leaves. The evidences from morphology showed the typical apoptotic hallmarks in cells undergoing programmed cell death. The results of terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and electrophoresis suggested that the special 3’-OH breaks and DNA fragmentation occurred in the chromatin DNA. On the other hand, Poly(ADP-ribose) polymerase (PARP), as an important nuclear protein and direct substrate of activated Caspase-3, was digested. All these evidences not only suggested that expression of caspase-3 gene could induce the programmed cell death in tobacco cells, but also implied that there is caspase-3 homolog and relative signal transduction pathway in plant cells.

The morphological change of nuclei is an important event during apoptosis process, and plays typical characteristics. However, we only know a little about this important event, because we can’t observed the morphological changes on the whole cell level. Because the membrane and organelles fractions were deprived from the carrot cell-free system, so we examined the nuclear changes, such as chromatin condensation, chromatin margination, and DNA Ladder, to analyze the molecular basis of the nuclear morphological changes during apoptosis. Transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase 6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We confirmed the results using whole- mount electron microscopy, and found that although the nuclear lamina was destroyed early, the nuclear lad-matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in maintaining the integrity of apoptotic nuclei bodies, and builds up a connection between these bodies and apoptotic nucleus.

Part II Effect of phosphocreatine on apoptosis induced in Xenopus cell-free system

During apoptotic process, cells can display many typical characteristic morphological and biochemical changes, which mainly owe to the cascade activation of caspases and the degradation of their substrates. Cytochrome c was released from the mitochondria of the apoptotic cells to the cytoplasm, where they binded with Apaf-1 and dATP or ATP, and activated procaspase-9. Activated caspase-9 cleaved and activated caspase-3, which in turn degraded its substrates specifically, such as procaspase-6 and DFF45. Procaspase-6 was cleaved and activated, degraded its substrate Lamin, leading to the apoptotic morphological changes. DFF45 was cleaved, releasing its binding nuclease, DFF40, to cleave the chromsome DNA into nucleosomal fragments, forming DNA Ladder after electrophoresis. Our lab established a success apoptotic system using Xenopus egg extracts cell-free system. And most of the typical apoptotic changes could be recovered in cell-free system based on Xenopus laevis egg extracts by addition of cytochrome c and exogenous mouse liver nuclei. Xenopus cell-free system was the classical model for cell cycle research, it can induce variety of cell cycle stages by the addition of ATP regenernation system (ATP, phosphocreatine, and phosphocreatine kinase). We herein report that the effects of phosphocreatine on apoptosis in this system. After phosphocreatine was introduced into this apoptosis inducing system, the apoptotic specific morphological changes were observed but not DNA fragmentation. The result of examining for activation of Caspase-3 suggested that the addition of phosphocreatine could buildup Caspase-3’s activation, which might be due to the addition of ATP-level in the inducing system. Western Blotting results showed that lamin A , C, were degraded as normal, which contributed to apoptotic morphological changes in nucleus, but the degradation of DFF45-like protein in egg extracts was inhibited. To exclude the possibility that ATP or acidic condition resulted from phosphocreatine might take, exogenous ATP or other organic acids were introduced into apoptosis inducing system. The result showed that they did not inhibit the formation of DNA fragmentation. On the other hand, the inhibition of phosphocreatine on formation of DNA fragmentation was time-dependent. This suggested that one or some factor(s) in cytosol was/were affected after phosphocreatine was added into, and this led to the inhibition of DNA fragmentation. Western Blotting evidences indicated that the degradation of DFF45-like protein in egg extracts was inhibited. Considering phosphocreatine could give out phosphate radical which might result in phosphorylation, LiCl, an inhibitor of Kinase, was added into system with phosphocreatine. The results suggested that DNA fragmentation and the degradation of DFF45-like protein recurred and the apoptotic morphological changes recover themselves. These indicated that DFF45-like protein was probably phosphorylated after addition of phosphocreatine, resulted in its resistance to caspase cleavage, and led to inhibition of DNA fragmentation.

Keywords: Cell-free system, nuclear reassembly, apoptosis, Lamin, Caspase3, phosphocreatine, DFF45.

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