用于研究植物质外体空间三维结构的树脂铸型技术

本文介绍了用于研究植物体质外体空间三维结构的树脂铸型技术。植物包含许多重要的质外体空间,如木质部管状分子的腔隙、与气孔相连的叶肉细胞间的通气系统、分泌腔等等。这种空间的三维结构可借助于扫描电子显微镜(SEM)研究。但问题是,用SEM直接观察不到一个组织或细胞切面内部的图像,因此,不能观察它们的全貌。通过运用树脂铸型技术,可以获得完整的组织或腔隙内部空间的铸型。反映管壁结构的各种形象被印在铸型的表面上,在SEM下可对质外体空间进行详细研究。树脂铸型技术在结构植物学的研究上有重要的意义。     

青心酮对妊娠高血压综合征胎盘血管内皮和平滑肌细胞内一氧化氮合酶的影响

本文对正常孕妇、妊娠高血压综合征（ＰＩＨ）患者和经青心酮（ＤＨＡＰ）治疗的ＰＩＨ患者等共２４例,应用组织化学分析方法观察胎盘血管内皮细胞（ＶＥＣ）和平滑肌细胞（ＶＳＭＣ）内一氧化氮合酶（ＮＯＳ）活性的变化。结果表明：正常孕妇胎盘ＶＥＣ和ＶＳＭＣ内ＮＯＳ活性较高;ＰＩＨ胎盘ＶＥＣ和ＶＳＭＣ内ＮＯＳ活性明显减弱,并伴有组织和细胞的形态学损伤;经ＤＨＡＰ治疗后的ＰＩＨ胎盘ＶＥＣ和ＶＳＭＣ细胞ＮＯＳ活性较未经ＤＨＡＰ治疗者明显增加,其组织和细胞损伤也减轻。本研究结果提示胎盘内ＶＥＣ和ＶＳＭＣ细胞的ＮＯＳ减少可能与ＰＩＨ的发生和／或发展有关,青心酮治疗ＰＩＨ的作用可能与ＤＨＡＰ促进胎盘ＶＥＣ和ＶＳＭＣ内一氧化氮（ＮＯ）合成有关。     

我国利用植物组织和细胞培养产生药用成分的研究概况

我国利用植物组织和细胞培养产生药用成分的研究概况王蜀秀,温远影,胡昌序（中国科学院植物研究所植物化学研究室,北京１０００４４）ＡＳＵＲＶＥＹＯＦＳＴＵＤＩＥＳＯＮＭＥＤＩＣＩＮＡＬＣＯＭＰＯ－ＮＥＮＴＳＰＲＯＤＵＣＥＤＢＹＰＬＡＮＴＴＩＳＳＵＥＡＮＤ...     

HDC细胞与MESPU—13细胞形成嵌合鼠能力的比较研究

ＥＳ细胞嵌合能力的强弱是人们利用ＥＳ细胞获得转基因小鼠时十分关心的问题。本言语通过囊胚显微注射法将１５个左右ＥＳ细胞注入Ｃ５７ＢＬ／６Ｊ品系小鼠３．５天囊胚的囊胚腔中观察嵌合鼠毛色嵌合情况。统计嵌合鼠的出生率;以及用葡萄糖磷酸异构酶（ＧＰＩ）电泳地检测ＥＳ细胞在嵌合鼠体内各种组织和器官的嵌合情况,对于ＨＰＲＴ缺陷（ＨＤＣ）细胞和ＭＥＳＰＵ－１３细胞的嵌合能力我们作了较详细的研究,结果表明ＭＥＳＰＵ     

ES细胞分化为血管样结构的机理探讨—TGF—β1在血管形成中的作用

本文利用小鼠ＥＳ细胞的拟胚一培养和三维胶原蛋白培养系统研究了外源ｒｈＴＧＦ－β１对ＥＳ细胞分化为血管样样结构的影响。结果发现,远方介培养中添加外源ｒｈＴＧＦ－β１或细胞经基因转染面有过度表达的ＴＧＦ－β１的ＥＳ－Ｔ６细胞分化为血管样结构的频率明显地受到抑制,相似于其亲本ＥＳ－５细胞的分化水平。在Ｉ型胶原三维培养系统中的单层ＥＳ－５细胞培液中添加ｒｈＴＧＦ－β１时,明显地促进ＥＳ－５细胞形成血管样结     

缺氧时肺动脉内皮细胞与肺动脉平滑肌细胞增殖的相互影响

血管内皮细胞和血管平滑细胞在结构和功能上关系密切,两者的相互在与血管舒缩笔血和壁结构。本文观察了培养的小牛肺动脉内皮细胞（ＰＡＥＣｓ）和肺动平滑肌细胞（ＰＡＳＭＣＳ）缺氧时在细胞增殖方面的相互影响。ＰＡＳＭＣＳ常氧条件培养基（ＣＭ）可使ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入降低约５８％,缺氧ＣＭ对ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入无明显的抑制作用;ＰＡＥＣＳ的常氧ＣＭ使ＰＡＳＭＳ的＾３Ｈ－ＴｄＲ掺入升高约６０     

金钱豹肾的组织学和微血管铸型的观察

为探讨金钱豹(Panthera pardus)肾的组织结构和微血管构筑特点,利用生物显微技术与微血管铸型技术及扫描电镜对金钱豹肾的组织结构和肾小球微血管构筑进行了观察。结果显示,金钱豹的肾皮质与髓质厚度比为1∶(1.5~3),肾近端小管上皮为单层立方细胞,胞体较大,细胞排列紧密,分界不清楚,其腔面有刷状缘,胞质嗜酸性;远端小管无明显的刷状缘,胞质弱嗜酸性,胞核多位于中央;集合管细胞排列紧密,管腔较大,上皮细胞呈矮柱状,胞质弱嗜酸性。金钱豹的肾小球多呈球形或卵圆形,直径为99~142μm,入球小动脉管径13~15μm,出球小动脉管径9.5~11.5μm。     

穿叶眼子菜茎叶结构及其通气组织发育解剖学研究

通气组织(aerenchyma)是植物薄壁组织内一些气室或腔隙的集合,对于水生及湿地植物体内的气体运输至关重要。该实验以沉水植物穿叶眼子菜为材料,利用石蜡切片技术,通过对茎的纵切面及横切面结构进行观察,从时间和空间上分析其茎、叶通气组织的发生过程。结果表明:(1)穿叶眼子菜的茎结构包括表皮、皮层及维管柱,通气组织发达,存在于内皮层与表皮之间;茎通气组织由距茎尖约0.6mm处开始形成,并成熟于约2.4mm处。(2)穿叶眼子菜的叶由表皮、皮层薄壁细胞及维管柱组成,其通气组织形成于靠近茎尖的第2~3片新生叶且仅形成于主叶脉。(3)穿叶眼子菜的茎和叶通气组织的发育过程相似,起初为排列致密的细胞团,然后由皮层细胞的分裂产生小的细胞间隙,随后的腔隙膨大过程涉及细胞的生长分裂及细胞降解,最终形成发达的通气组织。(4)穿叶眼子菜的通气组织发育过程可划分为实心期、形成期、膨大期、成熟期四个时期;不同时期茎通气组织的发达程度差异很大,实心期、形成期、膨大期和成熟期的孔隙度分别为0.54%、10.90%、27.61%和57.58%;但节处通气组织不发达,成熟期的节处孔隙度仅为3.62%。     

血啉甲醚在动脉组织的吸收分布特性

本研究观察血啉甲醚（ＨＭＭＥ）在血管平滑肌细胞（ＳＣＭ）、血管内皮细胞（ＥＣ）和动脉组织中的分布特点,旨在为建立防治血管成形术后再狭窄的光动力学疗法提供实验依据。将体外培养的ＥＣ和ＳＭＣ接种于２４忆培养板中,分组,培养液中加入ＨＭＭＥ,浓度为２０、４０、６０、８０、１００μｇ／ｍｌ,孵育１５、３０、６０、１２０、１８０、２４０ｍｉｎ后,采用荧光摄象分析法测定细胞内ＨＭＭＥ含量。以ＨＭＭＥ的孵育浓度     

壳菜果植物树脂道的发生、发育及分布的研究

经解剖观察,发现壳菜果（Ｍｙｔｉｌａｒｉａｌａｏｅｎｓｉｓ）植物初生结构能自然形成树脂道,而次生结构不能自然形成。树脂道原始细胞发生位置为离顶端１２０～１４０μｍ的区段上,与初生分生组织同时形成。以后原始细胞经历;（１）原始细胞分裂阶段;（２）树脂道发生阶段;（３）树脂道扩张阶段;（４）树脂道成熟阶段;（５）树脂道破毁阶段而完成其功能周期。壳菜果植物的树脂道以裂溶生方式发生,分布在维管束外侧的皮层中。此为金缕梅科各亚科系统位置研究提供重要证据     

Resin-casting: a method for investigating apoplastic spaces

A method has been developed in which liquid resin can be injected or infiltrated into spaces within a plant body, for example the lumens of vessels, fibers, and intercellular spaces. After polymerization of the resin, plant tissues are digested away with two solutions used in sequence: first, equal parts concentrated hydrogen peroxide and glacial acetic acid; second, concentrated sulfuric acid. Complete digestion renders three-dimensional casts of the spaces in the original tissue. These can be examined with scanning electron microscopy, light microscopy, or a dissecting microscope. Casts have such high fidelity and high resolution that details of pit canals, pit chambers, and perforation plates can be studied. Vessel casts over 15 cm long and revealing the details of several thousand constituent vessel elements have been obtained easily. Casts of the lumens of all cell types and of narrow intercellular spaces are obtained by prolonged infiltration with a low-viscosity resin solution. Alternatively, rapid, brief injection of the resin by vacuum produces casts predominantly of just those spaces that were open to the resin at the cut ends of the sample, for example the lumens of vessels and secretory ducts or the intercellular space network of an aerenchymatous parenchyma.     

A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast)

The intercellular spaces of sugarcane (Saccharum officinarum L.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation. It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 104 bacteria/mL extracted fluid) on N-poor medium containing 10% Suc (pH 5.5). This bacterium was identical with the type culture of Acetobacter diazotrophicus, a recently discovered N2-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air. Similar bacteria were observed in situ in the intercellular spaces. This demonstrates the presence of an N2-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium. The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop. This endogenous culture broth consisting of substrate and N2-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers. It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.     

The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.     

Apoplastic water fraction and rehydration techniques introduce significant errors in measurements of relative water content and osmotic potential in plant leaves

Relative water content (RWC) and the osmotic potential (π) of plant leaves are important plant traits that can be used to assess drought tolerance or adaptation of plants. We estimated the magnitude of errors that are introduced by dilution of π from apoplastic water in osmometry methods and the errors that occur during rehydration of leaves for RWC and π in 14 different plant species from trees, grasses and herbs. Our data indicate that rehydration technique and length of rehydration can introduce significant errors in both RWC and π. Leaves from all species were fully turgid after 1–3 h of rehydration and increasing the rehydration time resulted in a significant underprediction of RWC. Standing rehydration via the petiole introduced the least errors while rehydration via floating disks and submerging leaves for rehydration led to a greater underprediction of RWC. The same effect was also observed for π. The π values following standing rehydration could be corrected by applying a dilution factor from apoplastic water dilution using an osmometric method but not by using apoplastic water fraction (AWF) from pressure volume (PV) curves. The apoplastic water dilution error was between 5 and 18%, while the two other rehydration methods introduced much greater errors. We recommend the use of the standing rehydration method because (1) the correct rehydration time can be evaluated by measuring water potential, (2) overhydration effects were smallest, and (3) π can be accurately corrected by using osmometric methods to estimate apoplastic water dilution.     

Extraction of apoplastic sap from plant roots by centrifugation

A centrifugal method for extracting apoplastic sap from roots of lupin ( Lupinus angustifolius ) and pea ( Pisum sativum ) plants, and a method to analyse malic dehydrogenase in the sap using capillary electrophoresis, are described. Osmolality of apoplastic sap was relatively constant at relative centrifugal forces (RCFs) between 600 and 3000 g for lupin, and between 600 and 4000 g for pea. Electropherograms of a marker enzyme (malic dehydrogenase) and other components in apoplastic and symplastic saps revealed that contamination occurred at 7000 g . It is suggested that apoplastic sap expelled from plant roots at RCF between 600 and 3000 g is free from symplastic contamination, and is regarded as being of apoplastic origin. The proposed method was used to measure apoplastic pH changes in the plant roots in response to external pH, ammonium, nitrate and vanadate.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

植物根中质外体屏障结构和生理功能研究进展

综述了近10年来植物根中质外体屏障结构和功能的研究进展。质外体屏障指根中内、外皮层初生壁的凯氏带,或次生壁栓质化和木质化,以及植物体表角质层组成的保护组织,能隔绝水、离子和氧气不能自由进出植物体的屏障结构,具有保护植物体的生理功能。根中凯氏带的分子发育机理研究表明根内皮层类似哺乳动物上皮组织的保护作用。植物根中质外体保证内部各种生理代谢在稳定的内部环境中进行,是植物适应各种逆境的重要屏障结构。根中质外体屏障在植物适应干旱、洪涝灾害、离子胁迫和病虫害的侵袭等方面具有重要作用,在探索适应并修复极端生态环境的植物资源中有广阔的应用前景。     

Beyond iron-storage pool: functions of plant apoplastic iron during stress

Iron (Fe) is an essential micronutrient for plants, and its storage in the apoplast represents an important Fe pool. Plants have developed various strategies to reutilize this apoplastic Fe pool to adapt to Fe deficiency. In addition, growing evidence indicates that the dynamic changes in apoplastic Fe are critical for plant adaptation to other stresses, including ammonium stress, phosphate deficiency, and pathogen attack. In this review, we discuss and scrutinize the relevance of apoplastic Fe for plant behavior changes in response to stress cues. We mainly focus on the relevant components that modulate the actions and downstream events of apoplastic Fe in stress signaling networks.     

压力室测定根系导水率方法探讨

用压力室连续测定了玉米根系长压和降压过程的导水率,结果表明,降压过程湍 得的根系导水率显著大于用升压过程的,并且前者的相关系数大于后者,这种差异是由于这两个过程中质外体途径细胞壁空间充水量不同造成的,开始升压时,由于细胞壁空间含水量低,质外体途径阻力大,导致非结构阻力;随着压力的升高,细胞壁空间含水量增大,质外体途径导度增大,减小甚至可以消除非结构阻力,降压法可以使根系快速复水,消除传统方法因长时间复水所致根结构的改变。建议用降压法测定根系导水率。