体外受精研究:玉米和烟草性细胞融合的几种模式

分别用玉米(Zea mays L.)和烟草(Nicotiana tabacum L.)为材料,以单对细胞融合技术广泛开展了各种组合的性细胞的融合实验.重点观察了性细胞融合过程中细胞体积对细胞膜融合行为的影响.视频增差显微术结合细胞膜荧光标记技术显示,性细胞融合过程中受融合双方体积差异大小的调节,细胞膜的融合行为可归纳为3种基本模式;发现在体外受精条件下精细胞可以类似被吞噬的方式进入卵细胞,且其融合动作非常迅速.由此提出一种假设机制用以解释体内受精过程中雄性细胞质被排除的可能原因.     

细胞电融合法生产杂交瘤

细胞电融合过程包括使用交流电场使悬浮在缓冲液中的细胞排列成串,然后高压脉冲电流作用使细胞膜发生瞬时破裂,相邻细胞的细胞膜融合,继续受交流电场作用完成细胞融合。已经证明细胞电融合技术可用于杂交瘤生产,单克隆抗体生产以及细胞表面标记物细胞间的传递过程。     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

“体验细胞融合过程简易方法”实验教学的设计与组织

细胞膜具有一定的流动性,这是细胞融合、生物膜系统转化的基础。学生通过探究不同分子量不同体积分数的聚乙二醇(PEG)对细胞融合的影响,进而体验单克隆抗体的制备过程,加深对学科本质及学科思想的理解。     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇（PEG）诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

Mg2+浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等.其中,聚乙二醇(PEG)化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面.本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围.方法:以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg2+浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg2+浓度区间.结果:可以在原有的Hanks配方的基础上,调节Mg2+浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大.结论:Mg2+对细胞融合有一定影响,通过调节Mg2+浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案.     

Mg^2＋浓度对细胞融合效果的影响

目的：细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等。其中,聚乙二醇（PEG）化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面。本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围。方法：以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg^2＋浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg^2＋浓度区间。结果：可以在原有的Hanks配方的基础上,调节Mg^2＋浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大。结论：Mg^2＋对细胞融合有一定影响,通过调节Mg^2＋浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案。     

哺乳动物细胞电融合技术及其影响因素的研究进展

细胞电融合技术是指利用电脉冲促使两个或更多个不同的细胞融合在一起的过程。由于可以获得较高的融合率,而且可以在显微镜下定向诱导细胞融合和直接挑选杂种细胞,故电融合技术得到了广泛的应用。电融合技术对于哺乳动物杂种细胞的构建;哺乳动物四倍体发育的研究;以及胚胎细胞核移植都具有十分重要的意义。因此,人们对哺乳动物的电融合问题进行了大量的研究,现将这方面的研究进展做一综述。 (一)电融合技术的原理及方法 悬于平行电极之间的低电导率溶液中的细胞,在适当强度与持续时间的直流电脉冲的刺激下,其细胞膜可发生可逆电击穿,此时如两个具有膜微孔的细胞紧穿接触,可导致细胞质桥的形成,进而发生细胞融合。     

酿酒酵母细胞融合机制及交配信号通路的合成生物学应用

细胞融合是大多数真核生物发育中的一个基本生物过程。酿酒酵母作为真核生物基因组合成和转移的经典模式生物,其细胞融合机制不清楚,因此限制了它的合成生物学应用。在酿酒酵母的融合过程中,细胞对信息素做出反应,触发促分裂原活化的蛋白激酶（MAPK）级联反应以启动交配,随后细胞发生极化、细胞壁重塑、膜融合和核配。其中,研究可能的“融合酶”——受信息素调控的多跨膜蛋白（Prm1）为推动细胞融合可控性提供方向。酵母交配信号通路的合成生物学应用基于生物元件、生物装置与生物系统以及多细胞互作3个层次,本文分析了信息素诱导型启动子、G蛋白偶联受体、支架蛋白、转录因子、双稳态开关、调谐器、底盘细胞等在生物传感器及代谢工程等领域的应用。开发理性设计的模块化线路和优化交配途径来精确调控酵母交配的生理事件,对于细胞融合的人工可操纵性发展具有重要意义。     

细胞融合教学实验的改进

细胞融合技术是研究细胞遗传、基因定位、细胞免疫、病毒和肿瘤的重要手段。所谓细胞融合 ( cell fusion)又称体细胞杂交 ,是指用人工方法使两个或两个以上的体细胞融合成 1个双核或多核细胞 ,不经有性过程而得到杂种细胞。自细胞融合技术 ( Barski,1961)创建至今 ,依据融合过程采用的助融剂的不同可分为 :1)病毒诱导的细胞融合 ;2 )化学因子诱导的细胞融合 ;3 )电场诱导的细胞融合 ,此外还有激光诱导的细胞融合。在保证助融剂及操作技巧的前提下 ,细胞融合成功与否及融合率取决于实验所选择的亲代细胞。目前 ,普通高等院校和高等医学院校…     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

The First Record of Dynamics of Male-Female Nuclear Fusion in Viable Tobacco Central Cell During in Vitro Fertilization

In vitro double fertilization in tobacco (Nicotiana tabacum L. ) was carried out and one of its significant events, the dynamics of bisexual nuclear fusion in the viable central cell, was observed by video- enhanced microscopy for the first time. The observation revealed that after in vitro fertilization the male nucleus was first fused with one of the polar nuclei, then the other. The whole fusion process could be finished within two seconds. Morphologically the fusion was very similar to common protoplast fusion mediated by polyethylene glycol. It also went through a series of processes, namely touch, adherence, membrane fusion and content mixture. The male nucleolus moved closely towards the female one but no further fusion was recorded although eventually a big nucleolus was observed in the primary endosperm nucleus. The technique for in vitro fertilization and the observation of the nuclear fusion process may enable us to peep at the mechanism of male and female gamete fusion.     

The Saccharomyces cerevisiae PRM1 Homolog in Neurospora crassa Is Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Cell–cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Δ mutants show an ~50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an ~50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with ΔPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The ΔPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a ΔPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.     

In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion

The role of cell volume in regulating cell fusion was investigated by in vitro fertilization to elucidate mechanisms involved in double fertilization. The results revealed that in our model gamete fusion was more efficient than the fusion of somatic cells under the same conditions. The fusion of selected mesophyll protoplasts of different sizes and their fusion with chloroplasts demonstrated that cell volume ratio played a role in this process: as the ratio increased, fusion was more efficient and faster. When one of the cells was as small as a sperm, the formation of a round fusion product was faster. This might explain why gamete fusion was highly efficient in all in vitro germ cell fusion systems. This finding may also explain why sperm evolved as small cells. The results reported here will be useful for interpreting and evaluating data of in vitro fertilization experiments and for distinguishing gamete-specific characters. Received: 12 September 2000 / Revision accepted: 6 December 2000     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Behavior of the spermatozoon during sperm-blastomere fusion and its significance for fertilization (Saccoglossus kowalevskii: hemichordata)

Summary Association of spermatozoa with blastomeres removed from the egg envelope was studied in order to investigate sperm membrane behavior under circumstances unlike those attending fertilization of normal envelope-enclosed eggs. Under the altered circumstances all parts of the surface of the sperm cell were afforded the opportunity of meeting blastomere plasma membrane. Nevertheless, in each case the spermatozoon first became activated, an acrosomal tubule formed, and it was this organelle alone which fused and established continuity with the blastomere plasma membrane. This behavior of the spermatozoon with respect to the denuded blastomere parallels the behavior of the sperm cell with respect to the envelope-enclosed egg at fertilization. Since the sperm cell behaves so similarly in two situations which are so different with respect to what the spermatozoon encounters, this behavior is considered to reflect the inherent nature of the spermatozoon, and analysis of this behavior in relation to a blastomere is considered to be valid also in relation to an egg during fertilization. It is concluded that membrane fusion, involving the acrosomal tubule of an activated spermatozoon, is the only means by which gametic union is established and that these are obligatory rather than fortuitous features of sperm-egg association in Saccoglossus. It is suggested that these features are probably obligatory also in other species which exhibit this pattern of sperm-egg association.This investigation was supported by Research Grant HD-00007 from the National Institutes of Health, United States Public Health Service.     

离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。     

烟草中央细胞离体受精过程中雌雄核融合生活动态的记录

受精作用一直是植物生殖发育生物学研究的热点课题。近年发展尤为迅速。特别是诸如偏向受精等新概念的提出更进一步推动了对双受精作用的寻微探秘,日益显现出这一过程的精巧与复杂。但限于体内研究的局限性,对其中一些关键环节,如雌雄配子间的识别;配子融合过程中的相互作用;雄核在雌性细胞内迁移的动力学及雌雄核融合的时间进程与机制等仍知之甚少。离体受精操作及相关技术的建立［１～３］为探讨上述问题提供了新途径。我们在过去工作的基础上以烟草为材料进行了离体双受精研究,以视频增差显微观察系统首次记录到在生活状态下精核进…     

A novel in vitro system for gamete fusion in maize

Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of plant gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. In this study, we developed a bovine serum albumin （BSA）-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell and sperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG system, and it will be especially useful for the in vitro study of the calcium dynamics during plant fertilization.     

The Fusion of Male and Female Nuclei in Fertilization of Higher Plants

Studies on the fusion of male and female nuclei in fertilization of Helianthus an- nuus L., Triticum aestivan L., Gossypium hisutum L., Hosta caerulea Tratt., and Pinus tabulaeformis Carr. were made in the present work. The results are summarized as follows: 1. The essential process of the fusion of male and female nuclei during syngamy in four species of angiosperms studied may' be generalized as follows: (1) the male nucleus made contact with the female one, (2) followed by the fusion of nuclear membranes between the male and female nuclei. (3) then the despiralization of male spireme happened and male nucleolus made its appearance inside of the fertilized egg nucleus (4) the male chromatin dispersed and make its appearance indistinguishable from that of the female chromatin, (5) the male and female nucleoli fused together to form a larger nucleolus as a sign of completion of the fusion of the two nuclei. In the first mitotic division of the zygote there was only one common mitotic spindle. 2. The essential process of the fusion of egg and sperm nuclei during syngamy in a gymnosperm-Pinus tabulaeformis could also be outlined as follows: (1) the sperm nucleus made contact with the egg nucleue, (2) the fusion of nuclear membranes happened between the male and female nuclei, (3) the male and female ehromatins condensed to form two separate groups of chromatin threads together with the very apparent apperance of the male and female nucleoli at this stage, (4) the male and female chromosomes grouped respectively in their own spindles while both nucleoli disappeared, (5) then the two spindles fused together and all the chromosomes arranged to form a common equatorial plate, (6) finally two daugter nuclei resulted from the mitotic division. 3. Based on the facts that there were two different patterns of the fusion of male and female nuclei in fertilization discribed, all of these accounts are in general accord with the condition usually described that there are two types of fertilization, the pre- mitotic and postmitotie syngamy in higher plants. The type of angiosperm fertilization and the mechanism of promoting the zygote to divide after fertilization are discussed, and the nuclear fusion in sexual reproduction has been compared with that of somatic cell hybridization.