繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。     

海绵微生物生物活性物质的研究进展

海绵独特的摄食、滤食系统使其体内体表富集了大量的微生物,这些微生物能够产生多种结构新颖的生物活性物质,对海绵微生物的研究正在成为开发海洋药物资源的重要内容之一。本文概述了近十年来对海绵微生物生物活性物质的化学成分和生物活性的研究进展。     

海绵城市研究进展综述:从水文过程到生态恢复

在我国快速城市化进程中,不合理的规划建设加剧了城市洪涝、城市缺水、生态功能降低等诸多问题,受国外低影响开发理念启发,国内学者提出了与城市水文过程紧密结合的解决之道——"海绵城市"。在梳理海绵城市概念、内涵和发展现状的基础上,讨论了海绵城市建设与城市水文过程之间的作用关系,指出海绵城市研究涵盖水环境、水资源、水安全和水生态等众多领域且不断融合拓展。归纳了海绵城市的发展趋势,强调应更加遵循因地制宜原则,积极响应多尺度气候变化,科学认知城市水文过程并完善城市生态系统构建。提出了海绵城市建设优化框架,在理论上从单纯地关注水文过程转变到全方位倡导生态水文修复,在技术方法上重点融合生态水文途径改善城市水文空间体系,将生态恢复作为重要建设方式来提升城市应对灾害的弹性。     

海绵共附生真菌多样性及其次级代谢产物的研究进展

海绵由于其独特的生理结构、进食方式使其体内部聚集了大量的微生物,这些微生物产生了多种结构新颖的生物活性物质,因此海绵及其共附生微生物的研究成为了海洋药物研发的热点。就海绵中共附生真菌的分布情况,新技术的应用及其生物次级代谢产物的生物活性展开综述。     

海绵共生微生物研究中的分子技术

海绵共生微生物与海绵活性物质有紧密的关系,由于其大多不可培养,分子生物学技术成为海绵微生物研究的有效方法。本文重点介绍了海绵共生微生物研究中经常应用到的FISH、PCR、16S rRNA克隆测序、DGGE、RFLP等各种分子技术,对它们的特点及缺陷进行了分析与小结,希望有助于海绵共生微生物分子研究的进一步发展。     

NCS对离体萝卜子叶微体中酶活性及细胞超微结构的影响

研究了天然生物活性物质narciclasine(NCS)对离体萝卜子叶光下生长发育期间叶绿素含量变化、异柠檬酸裂解酶及羟基丙酮酸还原酶活性的影响;并对萝卜子叶细胞的超微结构变化进行了观察。结果表明,NCS明显抑制光下培养的离体萝卜子叶叶绿素含量及鲜重增加,对异柠檬酸裂解酶及羟基丙酮酸还原酶活性也显示出明显的抑制作用。电镜观察显示,NCS对蛋白体及脂质体的降解、叶绿体的发育也表现出强烈的抑制效应。NCS的各种抑制作用均随其浓度的增加而增加,而且高浓度的NCS(10^-5mol／L)基本上完全阻止了离体萝卜子叶的光下生长及其转绿。     

淡水海绵细胞类型

多孔动物(海绵动物)由于其独特的地位,在动物演化系谱上被列为一个侧支,又称侧生动物(Parazoa)。淡水海绵是供研究分化、形态发生及细胞之间通讯的一种极有用的动物。近代,据学者研究,在淡水生境中,淡水海绵更是环境质量的灵敏指示者。随着研究方法和电子显微镜等技术的进展,对淡水海绵细胞类型有更深入了解的必要。扁平细胞(Pinacocytes) 扁平细胞是海绵体表(外扁平细胞)和沿流水管道排列(内扁平细胞)的主要细胞,并形成基部的附着面(基扁平细胞)。此类扁平细胞形成单层扁平上皮。淡水海绵扁平上皮和高等动     

生物工程学报2002年第18卷总目录

第 1期综述肝细胞生长因子的分子生物学研究俞水亮　杨复华 ( 1)………………………………………………………………………………………………植物病原物无毒基因及其功能蔡新忠　徐幼平　郑　重 ( 5 )…………………………………………………………………………………………海绵生物活性物质及海绵细胞离体培养张骁英　赵权宇　薛　松　张　卫 ( 10 )……………………………………………………………………Ｎａ+ Ｈ+逆向转运蛋白和植物耐盐性任仲海　马秀灵　赵彦修　张　慧 ( 16 )…………………………………………………………………………     

植物细胞工程在十字花科作物种质创新中的研究进展

从胚拯救、小孢子培养、体细胞杂交、离体受精、体细胞无性系变异、染色体工程等六个方面综述了植物细胞工程技术在十字花科作物种质创新中的研究进展及应用,并对其发展前景进行了讨论。     

重要禾谷类植物转基因研究

与双子叶植物相比,禾谷类植物的离体培养和再生主要采用一些胚性组织,对它们的转基因研究发展相对比较缓慢。90年代以来,离体培养方面的经验被有效地融入DNA转移技术;通过筛选和再生少量的转化细胞,已得到了不少可育的小麦、水稻、玉米、大麦等禾谷类转基因植物。本文着重综述了禾谷类植物转化方法和选择体系方面的研究现状,同时讨论了该研究方向的未来发展趋势。     

Bioactive Natural Products from Papua New Guinea Marine Sponges

The discovery of novel natural products for drug development relies heavily upon a rich biodiversity, of which the marine environment is an obvious example. Marine natural product research has spawned several drugs and many other candidates, some of which are the focus of current clinical trials. The sponge megadiversity of Papua New Guinea is a rich but underexplored source of bioactive natural products. Here, we review some of the many natural products derived from PNG sponges with an emphasis on those with interesting biological activity and, therefore, drug potential. Many bioactive natural products discussed here appear to be derived from non‐ribosomal peptide and polyketide biosynthesis pathways, strongly suggesting a microbial origin of these compounds. With this in mind, we also explore the notion of sponge‐symbiont biosynthesis of these bioactive compounds and present examples to support the working hypothesis.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Farming Sponges to Supply Bioactive Metabolites and Bath Sponges: A Review

Sponges have been experimentally farmed for over 100 years, with early attempts done in the sea to supply “bath sponges”. During the last 20 years, sponges have also been experimentally cultured both in the sea and in tanks on land for their biologically active metabolites, some of which have pharmaceutical potential. Sea-based farming studies have focused on developing good farming structures and identifying the optimal environmental conditions that promote production of bath sponges or bioactive metabolites. The ideal farming structure will vary between species and regions, but will generally involve threading sponges on rope or placing them inside mesh. For land-based sponge culture, most research has focused on determining the feeding requirements that promote growth. Many sea- and land-based studies have shown that sponges grow quickly, often doubling in size every few months. Other favorable results and interesting developments include partially harvesting farmed sponges to increase biomass yields, seeding sexually reproduced larvae on farming structures, using sponge farms as large biofilters to control microbial populations, and manipulating culture conditions to promote metabolite biosynthesis. Even though some results are promising, land-based culture needs further research and is not likely to be commercially feasible in the near future. Sea-based culture still holds great promise, with several small-scale farming operations producing bath sponges or metabolites. The greatest potential for commercial bath sponge culture is probably for underdeveloped coastal communities, where it can provide an alternative and environmentally friendly source of income.     

Marine Sponges as Pharmacy

Marine sponges have been considered as a gold mine during the past 50 years, with respect to the diversity of their secondary metabolites. The biological effects of new metabolites from sponges have been reported in hundreds of scientific papers, and they are reviewed here. Sponges have the potential to provide future drugs against important diseases, such as cancer, a range of viral diseases, malaria, and inflammations. Although the molecular mode of action of most metabolites is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases have been reported. This knowledge is one of the key factors necessary to transform bioactive compounds into medicines. Sponges produce a plethora of chemical compounds with widely varying carbon skeletons, which have been found to interfere with pathogenesis at many different points. The fact that a particular disease can be fought at different points increases the chance of developing selective drugs for specific targets.     

Application of a MTT assay for screening nutritional factors in growth media of primary sponge cell culture

Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms. In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success. The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula. In 96-well plates, the effects of nutritional factors including glutamine, pyruvate, iron citrate, silicon, RPMI 1640, and Marine Broth 2216 on the viable cell density were examined in primary cell culture of S. domuncula 36 h after inoculation. Ferric iron (Fe(3+)) and pyruvate were found to significantly improve cell viability in a dose-dependent manner. Silicon and glutamine showed limited improvements at certain concentrations. The supplement of RPMI 1640 and Marine Broth 2216 did not increase cell viability. As a result, several improved media able to maintain higher cell viability in a short-term culture of primary sponge cells could be formulated.     

Species-Specific Association of the Cell-Aggregation Molecule Mediates Recognition in Marine Sponges

Reaggrcgation of dissociated cells of marine sponges, resulting in reformation of functional sponges, is a calcium-dependent process mediated by large, proteoglycan-like molecules termed aggregation factors (AF). During aggregation, species-specific sorting of cells is often observed. We purified and characterized AFs from three different sponge species and investigated their role in species-specific aggregation using novel approaches. The calcium-dependent association between purified AFs is species-specific in most combinations, as was shown in overlay assays and bead-sorting assays with AFs immobilized onto colored beads. Species-specific interactions of living cells and AF-beads resulted in incorporation of only homospecific AF-beads into reforming cell aggregates. Sequences from peptides obtained from the AF core proteins could all be aligned to the sequence of one species, the Microciona prolifera AFp3 core protein. In contrast to this similarity, major species-specific differences were seen in carbohydrate composition and in the response of AFs to specific carbohydrate-recognizing antibodies. In summary, our data point to a prominent role for the calcium-dependent association of AFs in recognition processes during aggregation. As this association of AFs occurs via carbohydrate -carbohydrate interactions, we speculate that the specificity of those interactions may be fundamental to recognition mechanisms required for regeneration of individuals from dissociated cells and for rejection of foreign material by sponge individuals.     

Initiation of an Aquaculture of Sponges for the Sustainable Production of Bioactive Metabolites in Open Systems: Example, Geodia cydonium

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state. Received September 16, 1998; accepted June 18, 1999     

Cellular Localization of Debromohymenialdisine and Hymenialdisine in the Marine Sponge <Emphasis Type="Italic">Axinella</Emphasis> sp. Using a Newly Developed Cell Purification Protocol

Sponges (Porifera), as the best known source of bioactive marine natural products in metazoans, play a significant role in marine drug discovery and development. As sessile filter-feeding animals, a considerable portion of the sponge biomass can be made of endosymbiotic and associated microorganisms. Understanding the cellular origin of targeted bioactive compounds from sponges is therefore important not only for providing chemotaxonomic information but also for defining the bioactive production strategy in terms of sponge aquaculture, cell culture, or fermentation of associated bacteria. The two alkaloids debromohymenialdisine (DBH) and hymenialdisine (HD), which are cyclin-dependent kinase inhibitors with pharmacological activities for treating osteoarthritis and Alzheimer's disease, have been isolated from the sponge Axinella sp. In this study, the cellular localization of these two alkaloids was determined through the quantification of these alkaloids in different cell fractions by high-performance liquid chromatography (HPLC). First, using a differential centrifugation method, the dissociated cells were separated into different groups according to their sizes. The two bioactive alkaloids were mainly found in sponge cells obtained from low-speed centrifugation. Further cell purifications were accomplished by a newly developed multi-step protocol. Four enriched cell fractions (C1, C2, C3, and C4) were obtained and subjected to light and transmission electron microscopy, cytochemical staining, and HPLC quantification. Compared to the low concentrations in other cell fractions, DBH and HD accounted for 10.9% and 6.1%, respectively, of dry weight in the C1 fraction. Using the morphological characteristics and cytochemical staining results, cells in the C1 fraction were speculated to be spherulous cells. This result shows that DBH and HD in Axinella sp. are located in sponge cells and mostly stored in spherulous cells.     

Sustainable Production of Bioactive Compounds by Sponges—Cell Culture and Gene Cluster Approach: A Review

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was—until recently—impossible to culture sponge cells in vitro. Also the predominant number of symbiotic bacteria proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the symbionts, a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases. From the consortium German Center of Excellence [BiotecMarin]. Dedicated to Dr. Paul J. Scheuer (University of Hawaii) who created the basis for the progress in the biomedical application of the bioactive potential of the marine environment.     

Efforts to develop a cultured sponge cell line: revisiting an intractable problem

Residents of the marine environment, sponges (Porifera) have the ability to produce organic compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Because of their sessile nature, the production of these bioactive compounds has been interpreted as a functional adaptation to serve in an important survival role as a means to counter various environmental stress factors such as predation, overgrowth by fouling organisms, or competition for limited space. Regardless of the reasons for this adaptation, a variety of isolated compounds have already proven to demonstrate remarkable anticancer, fungicidal, and antibiotic properties. A major obstacle to the isolation and production of novel compounds from sponges is the lack of a large, reliable source of sponge material. Sponge collection from the sea would be environmentally detrimental to the already stressed and sparse sponge populations. Sponge production in an aquaculture setting might appear to be an ideal alternative but would also be cost-ineffective and sponge growth is extremely slow. A third approach involves the development of a sponge cell culture system capable of producing the necessary cell numbers to harvest for research purposes as well as for the eventual commercial-scale production of promising bioactive compounds. Unfortunately, little progress has been made in this direction other than the establishment of temporary cultures containing aggregates and fragments of cells. One impediment toward successful sponge cell culture might be ascribed to a lack of published knowledge of failed methodologies, and thus, time and effort is wasted on continued reinvention of the same methods and procedures. Consequently, we have undertaken here to chart some of our unsuccessful research efforts, our methodology, and results to provide the sponge research community with knowledge to assist them to better avoid taking the same failed pathways.