空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温（４℃）、融合介质（０．５５ｍｏｌ／Ｌ甘露醇）并添加０．１％纤维素酶保存原生质体,７２ｈ内可以使约９４％细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心对亲本细胞之一去液泡,另一方面用电泳     

空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。     

细胞电融合技术在空间的发展潜力

以杂交瘤细胞生产和植物体细胞杂交为例，就发展空间细胞电融合技术问题的提出、技术关键、面临的挑战和在空间的发展潜力进行了分析。指出从国外研究结果来看，细胞电融合技术有可能发展成为未来的空间生物加工技术。     

微重力条件下细胞培养和组织工程研究进展

随着空间生命科学研究的发展,人们将细胞、组织培养技术与微重力环境相结合产生了组织工程研究的一个新领域——微重力组织工程。模拟微重力条件下细胞培养和组织构建研究表明,微重力环境有利于细胞的三维生长,形成具有功能的组织样结构,培养后的三维组织无论从形态上还是基因表达上都更接近于正常的机体组织。这种微重力对细胞的作用效应,将可能为未来组织工程和再生医学研究提供一条新途径。该文概述了近十年来国内外微重力组织工程相关研究的最新进展。     

微重力及模拟微重力对植物生长的影响

重力对地球上生物的生长、发育、代谢及繁殖等具有重要影响.植物细胞的重力敏感性已被众多研究所证明,在空间微重力环境或地面模拟微重力环境下,植物表现特殊的微重力反应.微重力或模拟微重力会对植物体生长产生一系列的影响.综述微重力及模拟微重力对植物生长的影响,并对近期这一领域的研究进行了概括.     

微重力环境影响植物生长发育的研究进展

微重力是最独特的空间环境条件之一,研究微重力对不同植物种类以及不同植物部位的影响是空间生物学的重要内容之一,对于建立生物再生式生命保障系统意义重大。生物再生式生命保障系统是未来开展长期载人空间活动的核心技术,其优势在于能在一个密闭的系统内持续再生氧气,水和食物等高等动物生活必需品,植物部件是生物再生式生命保障系统的重要组成部分。了解和掌握微重力对植物生长发育的影响,有助于采取有效的作业制度确保其正常生长发育和繁殖,是成功建立生物再生式生命保障系统的首要关键。该文就植物在空间探索中的地位和作用,地面模拟微重力的装置以及国内外有关微重力对植物的影响做一综述。现有的研究结果包括,未来长期的载人航天任务需要植物通过光合作用为生物再生式生命保障系统提供部分动物营养、洁净水以及清除系统中的固体废物和二氧化碳;三维随机回旋装置是目前地面上模拟微重力效应的主要装置之一,尤其适用于植物材料的长期模拟微重力处理;国内外有关微重力对植物影响的报道生理生化水平多集中在植物的生长发育和生理反应,比如表型变化或者与重力相关的激素或者钙离子的再分配,细胞或亚细胞水平主要有细胞壁、线粒体、叶绿体以及细胞骨架等,基因和蛋白质表达水平的研究对象主要为拟南芥。由于实验方法和材料之间的差异,微重力对不同植物或者植物不同部位在各个水平的影响效果并不一致,未来需要开展更多的相关研究工作。     

空间电融合的烟草原生质体再生植株分析

报告了在"神舟四号"飞船中通过电融合获得的烟草融合细胞,经地面培养获得再生植株。空间飞行样品再生愈伤组织的频率为地面对照的3倍多,而植株再生频率却比地面对照约低20%,约有32%的再生植株可能具有杂种性状。选取叶形状变化最为明显的H23号植株与黄花品种进行回交,回交一代的叶比原始材料宽,花的颜色与亲本黄花品种相同,表明空间微重力环境中融合的烟草原生质体能够再生植株,获得有繁殖能力的杂种。     

细胞电融合技术及最新进展

细胞电融合（cell electrofusion）是一种发展迅速的细胞工程技术,在细胞融合研究领域得到了最广泛的应用。细胞电融合利用细胞在相对电极之间的介电电泳,诱导细胞按特定方向排列,通过电极间产生的较高场强的电脉冲使相互接触的细胞发生电穿孔,进而发生电融合。融合后的细胞得到了不同细胞的遗传物质,具有新的遗传或生物特性。目前,细胞电融合技术对生物医学、农业等相关领域的研究具有非常重要的意义。本文介绍了细胞电融合技术及其最新研究动态,并简单介绍了本实验室在该领域的研究进展。     

模拟微重力诱导的细胞微丝变化影响COL1A1启动子活性

细胞骨架系统是细胞内的重力感受系统。已知微重力导致的细胞形态、功能、信号传导等多种变化均与细胞骨架系统变化有关,但微重力对相关基因调控的影响知之甚少。本研究以构建的基因工程细胞株（EGFP-ROS）为对象,以回转器模拟微重力效应,利用增强型绿色荧光蛋白（enhanced green fluorescence protein,EGFP）荧光半定量和细胞微丝荧光染色分析技术,探讨回转模拟微重力条件下,细胞微丝系统对Ⅰ型胶原α1链基因（collagen type Ialpha chain 1 gene,COL1A1）启动子活性的影响。空间飞行和回转模拟微重力后,细胞微丝解聚、张力纤维减少,表明微重力可降低细胞微丝结构的有序性,诱导细胞骨架重排。适合剂量的细胞松弛素B处理EGFP-ROS细胞诱导微丝骨架解聚,同时导致COL1A1启动子活性增加,细胞荧光强度增强,并呈现剂量依赖性。因此,一定程度的细胞微丝系统破坏将导致COL1A1启动子活性的增强,证明细胞微丝骨架系统参与了微重力对COL1A1启动子活性调节,且在微重力信号传导中起重要作用。     

空间植物科学与技术

2003年初,我国“神舟4号“载人试验飞船发射和回收成功,电视、电台和报纸都报道了我国科技人员在空间进行了植物细胞电融合.这仅是我国空间植物科学与技术研究的一小部分.……     

The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts

The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion.     

In vivo cell electrofusion

In vitro electrofusion of cells brought into contact and exposed to electric pulses is an established procedure. Here we report for the first time the occurrence of fusion of cells within a tissue exposed in vivo to permeabilizing electric pulses. The dependence of electrofusion on the ratio of applied voltage to distance between the electrodes, and thus on the achievement of in vivo cell electropermeabilization (electroporation) is demonstrated in the metastasizing B16 melanoma tumor model. The kinetics of the morphological changes induced by cell electrofusion (appearance of syncytial areas or formation of giant cells) are also described, as well as the kinetics of mitosis and cell death occurrence. Finally, tissue dependence of in vivo cell electrofusion is reported and discussed, since electrofusion has been observed neither in liver nor in another tumor type. Particular microenvironmental conditions, such as the existence of reduced extracellular matrices, could be necessary for electrofusion achievement. Since biomedical applications of in vivo cell electropermeabilization are rapidly developing, we also discuss the influence of cell electrofusion on the efficacy of DNA electrotransfer for gene therapy and of antitumor electrochemotherapy, in which electrofusion could be an interesting advantage to treat metastasizing tumors.     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41?±?9?% yield, while in isotonic buffer 32?±?11?% yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1?% in isotonic buffer to 10?±?4?% in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.     

Electrofusion and electroporation of plants

The utilization of electrofusion and electroporation techniques has had a major impact on the genetic manipulation of plants within the last decade. This review of the development of electrofusion and electroporation, as it applies to plants, highlights major developmental aspects of this technology. These include mechanisms for cell fusion, molecular exchange, and parameters that affect the efficiency of fusion and electroporation.     

Nuclear membrane fusion in electrofused mammalian cells

Fusion of nuclei was studied in electrofused cells using staining procedures and DNA flow cytometry. Homogeneous and heterogeneous electrofusion of Ehrlich ascites tumor cells. Muntjac cells and V79-S181 cells were performed in balanced-salt solutions at low temperature. Incubation of the cells subjected to electrofusion in fusion media for about 2 h was required to complete cell fusion and, in particular, nuclear membrane fusion. Under optimum electrofusion conditions it was found that fusion of nuclei is a very frequent event. Half of the fused cells (about 30 to 50% of the field-exposed cells) underwent nuclear membrane fusion. It is shown that the high frequency of nuclear membrane fusion in electrofused, unsynchronised cells resulted from intracellular dielectrophoresis occurring during cell alignment. In accordance with theory, maximum nuclear membrane fusion was observed using alignment fields of between 1 and 4 MHz (depending on the cell species), that is above the frequencies at which the plasmalemma capacity no longer shielded the cell interior from participation in the conduction process. In this frequency range a potential difference can be built up across the nuclear membrane leading to repositioning of the nuclei into the contact zone of the plasmalemmas of two attached cells. This intracellular dielectrophoresis apparently facilitated fusion of nuclei once intermingling of the plasma membranes had occurred. It was further demonstrated that exponentially growing cells showed higher cell fusion rates than cells taken from the unfed plateau phase. One, but not the only reason, might be the higher ATP content of exponentially growing cells compared to cells of the plateau phase. Addition of external ATP to plateau phase cells during electrofusion resulted, in accordance with this assumption, in an increase of fusion frequency, whereas ATP had apparently no effect on the fusion yield of exponentially growing cells. G1 cells obtained by mitotic selection after nocodazole-induced blockage in metaphase also showed higher cellular and nuclear membrane fusion yields than exponentially growing cells. Most importantly, it could be demonstrated both experimentally and theoretically that electrofusion of cells in a dielectrophoretically aligned chain is controlled by a simple law of probability resulting predominantly in fusion of two cells independent of the number of cells in the chain. The likelihood of fusion of various numbers of cells in a chain is given by the appropriate power of the probability of two-cell fusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions

Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.