植物细胞过氧化氢的测定方法

过氧化氢是重要的活性氧之一, 激素等发育信号和胁迫刺激可以诱导细胞内H2O2的产生和积累, 继而调控植物的气孔运动、生长发育、衰老和逆境应答等诸多生理过程。准确测定植物细胞内H2O2的含量及变化模式是系统研究H2O2信号转导及其生物学功能的一个关键技术。该文以拟南芥为实验材料, 介绍了目前植物细胞H2O2的主要测定方法, 包括激光共聚焦显微检测、紫外分光光度计检测和DAB组织染色, 在此基础上比较分析了上述方法在灵敏度、检测范围、定量、成本以及耗时等方面的差异, 为相关研究选择合适的H2O2检测技术提供参考。     

过氧化氢在水杨酸诱导的蚕豆气孔关闭中的作用

许多植物病原菌可通过气孔进行叶片组织,因此减小气孔开度有利于提高植物的抗性,我们通过表皮条分析和激光扫描共聚显微镜得到的证据表明在保卫细胞中过氧化氢可能是水杨酸信号的中间环节。SA可以浓度依赖的方式诱导气孔关闭（图1A）,H2O2也有类似的作用（图1B）。100μmol/L的水杨酸诱导的气孔关闭作用可明显地被20U/ml的过氧化氢酶或10μmol/L的Vc逆转,但CAT和Vc单独处理时诱导气孔开放的作用很微弱,单细胞中基于荧光探针DCFH的时间进程实验表明直接外加（图版Ⅰ）或显微注射100μmol/L的SA均可诱导保卫细胞中H2O2产生,但以显微注射双蒸水作为对照时对DCFH荧光无影响（图版Ⅱ）,这些结果暗示了植物被病原菌感染时可能通过产生H2O2导致气孔关闭而阻止病原菌继续通过气孔侵入。     

内窥式激光共聚焦显微镜

为了能在活体内进行实时的细胞尺寸水平的共聚焦观测,科研工作者开展了大量的将激光共聚焦显微镜和内窥镜技术相结合的内窥式激光共聚焦显微镜的研究.本文主要列举了一些国外典型的内窥式激光共聚焦的结构及性能参数,介绍了我们在这方面取得的成果.我们建立的结构的横向分辨率为3.3 μm,轴向分辨率为16.6 μm.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

利用转盘共聚焦显微镜进行快速实验的新方法

转盘共聚焦显微镜是快速激光共聚焦显微镜的一种,与传统的激光共聚焦显微镜相比具有一些相同点,也有其特有的优势。本文主要介绍转盘共聚焦显微镜的基本原理及如何利用转盘共聚焦显微镜进行快速实验及应用实例,并与传统激光共聚焦显微镜进行比较。转盘共聚焦显微镜具有速度快、灵敏度高、对样品光损伤和光淬灭程度低、操作灵活简单,是随着实验技术发展使用越来越广泛的实验仪器。     

百合花粉细胞中钙离子的荧光测定法1

以川百合花粉为材料,利用孵育法在花粉原生质体中,特别是利用低温装载法在完整花粉粒中,成功地载入酯化形式的钙离子荧光探针fluo-3AM.利用激光共聚焦显微技术对花粉细胞质中游离钙离子的分布特点进行研究的结果表明,花粉萌发初期细胞质内游离钙离子呈极性分布,萌发沟附近的钙离子浓度明显高于其他部位,萌发孔附近最高,花粉细胞核中较低.文中对低温装载法的可行性进行了讨论.     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

活性氧不敏感型拟南芥的突变体对H2O2的响应

检测拟南芥ros突变株对H2O2响应的结果表明,此种突变体对H2O2有较强的耐受性,表现为气孔开度对H2O2不敏感和H2O2胁迫时的膜脂过氧化水平较低。采用激光扫描共聚焦显微术(LSCM)并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞的结果显示,突变体内荧光强度比野生型拟南芥低,暗示此种突变体消除H2O2的能力可能有提高,从而可增强植株抗氧化胁迫的能力。     

百合花粉细胞中钙离子的荧光测定法

以川百合花粉为材料,利用孵育法在花粉原生质体中,特别是利用低温装载法在完整花粉粒中,成功地载入酯化形成的钙离子荧光探针fluo-3AM,利用激光共聚焦显微技术对花粉细胞质中游离钙离子的分布特点进行了研究的结果表明,花粉萌发初期细胞质内游离钙离子呈极性分布,萌发沟附近的钙离子浓度明显高于其他部位,萌发孔附近最高,花粉细胞核中较低,文中对低温装载法的可行性进行了讨论。     

过氧化氢在水杨酸诱导的蚕豆气孔关闭中的作用

许多植物病原菌可通过气孔进入叶片组织,因此减小气孔开度有利于提高植物的抗性。我们通过表皮条分析和激光扫描共聚显微镜得到的证据表明在保卫细胞中过氧化氢可能是水杨酸信号的中间环节。SA可以浓度依赖的方式诱导气孔关闭（图1A）,H2O2也有类似的作用（图1B）。100μmol/L的水杨酸诱导的气孔关闭作用可明显地被20U/ml的过氧化氢酶或10μmol/L的Vc逆转,但CAT和Vc单独处理时诱导气孔开放的作用很微弱。单细胞中基于荧光探针DCFH的时间进程实验表明直接外加（图版I）或显微注射100μmol/L的SA均可诱导保卫细胞中H2O2产生,但以显微注射双蒸水作为对照时对DCFH荧光无影响（图版II）。这些结果暗示了植物被病原菌感染时可能通过产生H2O2导致气孔关闭而阻止病原菌继续通过气孔侵入。     

ABA诱导蚕豆气孔保卫细胞H2O2的产生

以蚕豆叶片下表皮为材料,将荧光探针ＨＰＴＳ导入蚕豆气孔保卫细胞内,利用荧光光谱和激光共聚焦显微技术,检测了ＡＢＡ诱导蚕豆气孔关闭过程中Ｈ２Ｏ２的产生。结果表明,ＡＢＡ和１００μｍｏｌ／Ｌ的Ｈ２Ｏ２可以迅速猝灭ＨＰＴＳ的荧光,ＣＡＴ几乎可完全阻止９μｌ０．２ｍｍｏｌ／Ｌ的ＡＢＡ引起荧光猝灭。因此,ＡＢＡ可以诱导气孔保卫细胞产生Ｈ２Ｏ２,其产生的强度和速度与ＡＢＡ的浓度直接有关,并且推测：这种Ｈ２Ｏ２产生可能是ＡＢＡ诱导气孔关闭过程中信号转导链的一个中间成分。     

Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

Hydrogen Peroxide and the Proliferation of Bhk-21 Cells

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H₂O₂, such as the superoxide dismutase mimic, copper II—(3,5-diisopropylsalicylate)₂, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 μM exogenous H₂O₂ to the culture medium. Under these conditions, however, intracellular levels of H₂O₂ are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H₂O₂ and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H₂O₂, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to “trigger” their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H₂O₂     

Carbon Monoxide-induced Stomatal Closure Involves Generation of Hydrogen Peroxide in Vicia faba Guard Cells

Here the regulatory role of CO during stomatal movement In Vicla faba L. was surveyed. Results Indicated that, like hydrogen peroxide (H2O2), CO donor Hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations, showing the first time that CO and H2O2 exhibit the similar regulation role in the atomatal movement. Moreover, our data showed that ascorbic acid (ASA, an important reducing substrate for H2O2 removal) and diphenylene iodonium (DPI, an inhibitor of the H2O2-generating enzyme NADPH oxidase) not only reversed stomatal closure by CO, but also suppressed the H2O2 fluorescence induced by CO, implying that CO induced-atomatal closure probably involves H2O2 signal. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO specific synthetic inhibitor ZnPPIX, ASA and DPI reversed the darkness-induced stomatal closure and H2O2 fluorescence. These results show that, perhaps like H2O2, the levels of CO in guard cells of V. faba are higher In the dark than in light, HO-1 and NADPH oxidase are the enzyme systems responsible for generating endogenous CO and H2O2 in darkness respectively, and that CO is involved in darkness-induced H2O2 synthesis in V. faba guard cells.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

干旱条件下大豆叶片H_2O_2代谢变化及其同抗旱性的关系

干旱条件下大豆叶片H_2O_2含量增加,AsA POD与 GR活性均表现“先上升后下降”的趋势。叶片AsA与GSH含量均随干旱时间的延长而逐渐下降,而PPOD活性则持续增加。抗旱性较强的小粒大豆品种7605在干旱条件下能维持较强的 H_2O_2清除能力,H_2O_2累积较少。