2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation

Suspensions of thymocytes from young rats were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which resulted in a sustained increase in cytosolic free Ca2+ concentration followed by DNA fragmentation and loss of cell viability. Both the Ca2+ increase and DNA fragmentation were prevented in cells treated with the inhibitor of protein synthesis, cycloheximide, and DNA fragmentation and cell killing were not detected when cells were incubated in a "Ca2+-free" medium or pretreated with high concentrations of the calcium probe, quin-2 tetraacetoxymethyl ester. These results indicate that TCDD can kill immature thymocytes by initiating a suicide process similar to that previously described for glucocorticoid hormones.     

氧化应激在镉致大鼠肝细胞凋亡中的作用

 【目的】探讨氧化应激在镉诱导大鼠肝细胞凋亡中的作用。【方法】采用两步灌流法获得大鼠肝细胞,经过24 h培养,用醋酸镉处理细胞,或者Z-VAD-fmk、NAC和镉共同处理细胞。应用MTT法检测细胞存活率,流式细胞仪检测细胞凋亡率、细胞内ROS水平和线粒体膜电位,分光光度法检测caspase-3活性、GSH和MDA含量。【结果】结果表明,肝细胞暴露于浓度为2.5、5、10 μmol•L-1的镉后,细胞相对存活率显著下降（P＜0.01）,凋亡率显著或极显著升高（P＜0.05或P＜0.01）,呈剂量-效应关系;2.5和5 μmol•L-1镉组在1.5 h之前导致细胞内ROS水平显著或极显著升高（P＜0.05或P＜0.01）;镉暴露可使肝细胞Δψm显著或极显著降低（P＜0.05或P＜0.01）;NAC能够显著减少镉引起的凋亡细胞数量和极显著降低凋亡率（P＜0.01）,可以显著降低单独镉暴露组ROS水平升高和阻止ΔΨm降低（P＜0.05）;细胞内GSH含量12 h时随镉剂量增高而降低,部分剂量组极显著低于对照组（P＜0.01）,24 h时随剂量增高而升高,部分剂量组显著或极显著高于对照组（P＜0.05或P＜0.01）;细胞内MDA含量随着镉浓度的增大而升高,10 μmol•L-1剂量组MDA的含量显著高于对照组（P＜0.05）;未见caspase-3活性升高,caspase抑制剂Z-VAD-fmk对镉致细胞凋亡无影响。【结论】醋酸镉致肝细胞凋亡与其引起ROS产生并导致氧化损伤的非caspase途径有关。     

Cytosolic-free calcium transients in cultured vascular smooth muscle cells: microfluorometric measurements

Microfluorometric recordings were made of changes in the concentration of cytosolic-free calcium in cultured rat vascular smooth muscle cells treated with quin 2, an intracellularly trapped dye, under several conditions. Nitroglycerin decreased calcium in both the presence and absence of extracellular calcium and strongly and progressively decreased the extent of transient increases in calcium induced by repeated applications of caffeine in the absence of extracellular calcium. Therefore nitroglycerin probably decreases cytosolic-free calcium by accelerating the extrusion of calcium through the sarcolemmal membrane.     

Oscillations of cytosolic sodium during calcium oscillations in exocrine acinar cells

In acinar cells from rat salivary glands, cholinergic agonists cause oscillations in cytoplasmic free calcium concentration, which then drive oscillations of cell volume that reflect oscillating cell solute content and fluid secretion. By quantitative fluorescence ratio microscopy of an intracellular indicator dye for sodium, it has now been shown that large amplitude oscillations of sodium concentration were associated with the calcium and cell volume oscillations. Both calcium and sodium oscillations were dependent on the continued presence of calcium in the extracellular medium and were abolished by the specific sodium-potassium adenosine triphosphatase inhibitor ouabain. Thus, calcium oscillations in salivary acinar cells, by modulating the activities of ion transport pathways in the plasma membrane, can cause significant oscillations of monovalent ions that may in turn feed back to regulate calcium oscillations and fluid secretion.     

Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent beta-mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.     

Isolated adrenal cells: adrenocorticotropic hormone, calcium, steroidogenesis, and cyclic adenosine monophosphate

Corticosterone production by isolated adrenal cells in response to adrenocorticotropic hormone is reduced when the cells are incubated in a medium that contains no calcium. This reduction is associated with an equal reduction of accumulation of cyclic adenosine monophosphate. Production of corticosterone and accumulation of cyclic adenosine monophosphate are increased when the calcium concentration in the medium is increased (from zero to 7.65 millimolar). This is in contrast to the situation in "subcellular membrane fragments" of adrenal tissue where high calcium in the medium (> 1.0 millimolar) inhibits cyclic adenosine monophosphate accumulation. We propose that adenyl cyclase in the intact plasma membrane is located in a compartment wherein calcium concentration is low and remains unaffected by the concentration of calcium in the extracellular space. It is proposed that, as the concentration of calcium in the incubation medium is increased from zero to 7.65 millimolar, the strength of the signal generated by the interaction of adrenocorticotropic hormone with its receptor and transmitted to the adenyl cyclase compartment is proportionately increased.     

枸杞多糖对四氯化碳诱导建鲤原代肝细胞损伤的保护作用研究

研究中药枸杞多糖(Lycium barbarum polysaccharide,LBP)对四氯化碳(CCl4)诱导的建鲤(Cyprinus carpiovar Jian)肝细胞急性损伤的保护作用。利用8 mmol/L四氯化碳构建建鲤肝细胞损伤模型,用不同浓度的枸杞多糖处理肝细胞,设空白对照组、枸杞多糖对照组(0.4 mg/mL)、模型组(CCl4)、预防组(CCl4处理前加枸杞多糖孵育,0.1、0.2和0.4 mg/mL)、治疗组(CCl4处理后加枸杞多糖孵育,0.1、0.2和0.4 mg/mL)和预防治疗组(枸杞多糖孵育后经CCl4处理再经枸杞多糖孵育,0.1、0.2和0.4 mg/mL)。12 h后,收集细胞和细胞培养液,然后测定谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、细胞活力、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、免疫球蛋白M(IgM)及药物代谢酶细胞色素P450 2E1(CYP2E1)等生化指标。研究结果表明:CCl4处理后可以显著增加GPT、GOT、LDH、MDA、TNF-α、IL-1β、IgM及CYP2E1的含量,降低SOD活性和细胞活力;枸杞多糖处理组可不同程度地抑制上述生化指标的变化,其中预防组及预防治疗组对于转氨酶(GPT、GOT)、LDH、MDA、细胞因子(TNF-α、IL-1β)、IgM及CYP2E1等活性的升高皆有显著抑制作用,同时显著提高SOD酶活性及细胞活力,且呈剂量依赖性;而治疗组仅对LDH、TNF-α及CYP2E1有显著的抑制作用。综上结果,认为枸杞多糖对四氯化碳致建鲤肝损伤具有一定保护作用,可以应用于鱼类肝损伤的防治。     

鸡柔嫩艾美耳球虫对宿主细胞凋亡的影响

为了研究柔嫩艾美耳球虫入侵与宿主细胞之间的相互关系,用纯化的柔嫩艾美耳球虫子孢子与MDBK细胞共培养,使球虫子孢子入侵MDBK（Madin-Darby bovine kidney）细胞,用终浓度6%乙醇进行凋亡诱导,通过Annex-in V/PI双荧光染色法和流式细胞术（FCM）对MDBK细胞凋亡水平进行检测。试验结果证明子孢子入侵细胞后,有子孢子入侵的细胞凋亡诱导显著被抑制（P〈0.05）,而未有子孢子入侵的细胞相继被凋亡诱导剂诱导凋亡。进一步揭示柔嫩艾美耳球虫子孢子具有独特的机制来延长宿主细胞的存活时间来确保它们能在细胞中存活并发育。     

鲤鱼肝细胞的原代培养研究

[目的]探讨鲤鱼肝细胞原代培养的最佳条件。[方法]通过对鲤鱼的肝脏细胞进行原代培养,分析不同培养基、温度、pH、胰蛋白酶浓度、生长时间对鲤鱼肝细胞生长状况的影响,并测定肝细胞活力。[结果]鲤鱼肝细胞培养的最佳条件为:温度在25℃左右,pH7.4,胰蛋白酶浓度0.250%,消化时间40 min,培养基为M199。分离肝细胞的平均存活率>85%,每克肝平均可获得3.8×106个分离细胞,在细胞活力及数量上达到最佳平衡。[结论]该研究可为建立稳定的毒理学和药理学试验模型奠定基础。     

The relationship between charge movements associated with ICa and INa-Ca in cardiac myocytes

Ventricular myocytes exhibit a nifedipine-sensitive inward calcium current (ICa) and contracture when they are voltage clamped from -40 to 0 millivolt in the presence of caffeine and in the absence of extracellular sodium. However, upon repolarization they fail to relax because neither the sarcoplasmic reticulum nor the sodium-calcium exchange can reduce intracellular calcium. Sudden application of extracellular sodium during the contracture (but after repolarization) causes immediate relaxation and activates a transient inward sodium-calcium exchange current (INa-Ca), whose peak slightly precedes mechanical relaxation. The total charge carried by the nifedipine-sensitive ICa is twice the total charge carried by the transient inward INa-Ca. Assuming an exchange stoichiometry of three sodium to one calcium, these results indicate that all the calcium entering the cell during the initial depolarization is extruded by the sodium-calcium exchange.     

维生素C·E对草鱼片贮藏品质的影响

[目的]研究维生素C、E处理对草鱼片贮藏品质的影响。[方法]不同处理草鱼片在冷藏、微冻和冷冻2 d后,测定其失重率、挥发性盐基氮、pH值、白度。[结果]维生素E处理的持水性好于维生素C处理,但均高于空白;维生素C、E处理对鱼片的pH值影响较小,比较接近于空白样品;在3种处理中,维生素C处理的样品挥发性盐基氮值最低;维生素C、E对冷藏、微冻状态下的草鱼片具有良好色泽保护作用。[结论]维生素C对冷藏、微冻状态下的草鱼片具有良好的降低水产品腐败和色泽保护作用;维生素E对冷藏、微冻状态下的草鱼片具有良好色泽保护作用。     

镉对大鼠原代培养肝细胞毒性的研究

为探讨镉对原代大鼠肝细胞的毒性作用。用二步灌流法获得大鼠肝细胞,肝细胞暴露于浓度为2.5、5.0、10.0、20.0μmoL/L的醋酸镉中12、24 h,应用四甲基偶氮噻唑蓝(MTT)比色法检测醋酸镉对肝细胞存活率的影响及培养上清液中乳酸脱氢酶(LDH)的活性变化。结果表明,肝细胞暴露于浓度为2.5、5.0、10.0、20.0μmoL/L的醋酸镉中,细胞相对存活率显著下降(P0.01),LDH的释放量增加,且具有剂量效应关系。提示一定浓度的醋酸镉可引起肝细胞的毒性损伤。     

铜和锌对小麦胚芽愈伤组织维生素E积累的影响

[目的]考查培养基中铜元素和锌元素的含量对小麦胚芽愈伤组织维生素E积累的影响。[方法]通过建立维生素E积累和细胞生长的数学模型,描述小麦胚芽愈伤组织的维生素E积累和细胞增长。以次生代谢产物维生素E积累最多为目标,通过试验优化确定铜锌含量的最佳组合。[结果]结果表明,当B5培养基中ZnSO4.7H2O为2.0mg/ml;CuSO4.5H2O为0.1mg/ml时,有利于维生素E积累。维生素E积累的动力学模型的拟合度为97.53%,细胞干重的动力学模型拟合度为95.60%,非线性关系良好。[结论]微量元素铜和锌对小麦胚芽愈伤组织维生素E积累有影响,铜元素较锌元素的影响显著,且低浓度的铜锌之间有协同作用;随着浓度增大,抑制作用逐渐增强。     

抑制钙信号转导对柔嫩艾美耳球虫入侵细胞的影响

 【目的】探讨柔嫩艾美耳球虫（Eimeria tenella）入侵细胞与钙信号转导之间的关系,揭示鸡柔嫩艾美耳球虫病的发生机制。【方法】采用体外狗肾细胞（madin-darby canine kidney cell,MDCK细胞）培养技术,检测了细胞外缺钙、Ca2+内流阻断剂（硝苯地平）和钙调蛋白抑制剂（三氟拉嗪）对E. tenella子孢子入侵率的影响,并测定了培养细胞上清液中乳酸脱氢酶（LDH）和细胞活性。【结果】E. tenella子孢子入侵细胞的抑制率随着细胞外Ca2+浓度的降低而升高。钙离子浓度降低到600 μmol•L-1时,入侵率（23.33%）均极显著低于对照组（P＜0.01）;细胞外无钙时,入侵抑制率高达53.18%;硝苯地平和三氟拉嗪均能极显著抑制子孢子的入侵（P＜0.01）,其中10 μmol•L-1的硝苯地平和50 μmol•L-1三氟拉嗪分别对子孢子的入侵抑制率达71.41%和97.13%,二者合用入侵抑制率可达98.59%。在E. tenella子孢子入侵细胞的过程中,MDCK细胞的活性均在90%以上,与对照组差异不显著（P＞0.05）,接种E. tenella子孢子的MDCK细胞培养液中LDH的活性与未接种的活性差异不显著（P＞0.05）。【结论】细胞外钙缺乏、钙通道阻断剂硝苯地平和钙调蛋白抑制剂三氟拉嗪对E.tenella子孢子入侵MDCK细胞均有抑制作用,但子孢子入侵对MDCK细胞的活性无明显影响。     

红平菇菌株H1的培养特性与营养成分分析

分别在PDA培养基、马铃薯半组合培养基和MEA培养基上研究了红平菇菌丝体的培养特性,并对栽培后获得的红平菇子实体的各种营养成分进行了分析。培养特性的研究结果表明,红平菇在不同的培养基上宏观和微观培养特性各有不同,但菌体在3种培养基上都能够产生细胞外酚氧化酶,说明红平菇是一种白腐菌。菌丝在PDA和马铃薯半组合培养基上生长旺盛,白色,绒毛状至絮状,略显粉状,在马铃薯半组合培养基上呈现出明显的环纹;在MEA培养基上生长较弱,气生菌丝体较稀疏。菌体在3种培养基上产生的微观特征包括多数为节状分隔少数为简单分隔的薄壁菌丝、膨大的头状囊状体、很多八面体形和圆柱形的晶体等结构。对红平菇子实体营养成分分析的结果表明,红平菇是一种高蛋白低脂肪的健康食品,富含多种人体必需和非必需的氨基酸、多种维生素和矿物元素。其中新鲜子实体中水分含量为90．16％,自然风干的子实体中水分含量为6．36％。自然风干的子实体中粗脂肪的含量为2．63％;粗蛋白含量为24．69％;总酸度仅为0．187 3％;总灰分含量为9．43％;还原糖含量为0．507％;氨基酸总量为17．6％,其中含有人体必需的氨基酸包括异亮氨酸、亮氨酸、苯丙氨酸、赖氨酸、苏氨酸、缬氨酸、蛋氨酸(由于采用酸水解的方法分析氨基酸的成分与含量,因此没有检测出色氨酸),还含有其他10种非必需氨基酸;每100 g自然风干的子实体粉碎样品中含VB10．032 mg、VB20．469 mg、VC0．20 mg、VE701．16 mg;每100 g自然风干的子实体粉碎样品中含大量元素钾1 292 mg、磷1 206 mg、钠338．06 mg、钙151．44 mg、镁262．25 mg;每100 g自然风干的子实体粉碎样品中含微量元素锌16．1 mg、铁10．89 mg、铜1．53 mg、锰1．46 mg。     

有毒有机物影响DNA酶解和抗生素抗性基因横向迁移

进入环境中的有毒有机物种类较多、分布广泛。大量生命遗传物质DNA与有毒有机物共存于污染环境中,由于抗生素滥用,一些DNA上携带着抗生素抗性基因(ARGs);有毒有机物和ARGs复合污染严重危害着生态安全和人群健康。有毒有机物如何影响DNA酶解和ARGs迁移,这已成为环境领域研究的热点之一,近些年来,该领域研究取得一些重要进展。有毒有机物可通过共价作用、非共价作用、剪切作用与DNA结合,进而改变DNA分子结构、影响DNA酶解;有毒有机物也可通过改变DNA降解酶活性或占据DNA上降解酶的酶切位点,进而影响DNA酶解过程。环境中ARGs横向迁移主要有接合、转导、转化三种方式,有毒有机物影响ARGs横向迁移的机制主要包括:有毒有机物调控可动遗传因子、引起细胞SOS反应、胁迫细胞形成自然感受态、影响生物膜形成、改变细胞膜通透性、与胞外质粒形成加合物。进入细胞后的有毒有机物如何作用于ARGs的复制和表达,这应是未来深化有毒有机物影响ARGs横向迁移机制的一个关键所在。     

葡萄皮多酚调节LC3-Ⅱ表达及抑制视网膜色素上皮细胞损伤

研究葡萄皮多酚类化合物是否具有保护RPE细胞免受对脂褐素损伤的作用,并且探讨这种保护作用是否与维持自噬活性相关。脂褐素沉积是细胞衰老标志之一,当脂褐素在视网膜色素上皮细胞(RPE)中累积到一定水平,将增加年龄相关性黄斑变性(AMD)的风险。但是,脂褐素在RPE细胞内的作用机制尚不清晰。目前尚无有效预防、治疗AMD的手段,通过抗氧化剂清除自由基,维持细胞自噬活性可能会成为预防AMD的新策略。采用化学合成脂褐素主要荧光基团Nretinyledin-N-retinylethanolamin(A2E)建立RPE细胞累积脂褐素模型,用共聚焦显微镜和液相色谱-质谱联用检测A2E在RPE细胞中累积。细胞活力及凋亡检测结果表明A2E对细胞活性具有抑制作用。Western blotting结果表明A2E提高自噬相关蛋白LC3-Ⅱ的累积,并且自噬阻断剂bafilomycin A1和A2E共同处理RPE细胞条件下LC3-Ⅱ表达量较单独用A2E处理条件下没有显著性差异,说明A2E能够阻断自噬流。此外,葡萄皮提取物能够减少A2E诱导的LC3-Ⅱ累积,提示葡萄皮多酚化合物对自噬流的保护作用。并且,葡萄皮提取物能够抑制A2E诱导RPE细胞凋亡,这种抑制作用在一定程度上归因于对自噬流的保护作用。     

Platelet-mediated cholesterol accumulation in cultured aortic smooth muscle cells

Cholesterol accumulates within smooth muscle cells and macrophages in atherosclerotic lesions, thereby contributing to the progressive enlargement of these lesions. The mechanism of this cellular accumulation of cholesterol is not known. The possibility that platelets may have a role in the cellular cholesterol accumulation that occurs during atherogenesis was investigated. Incubation of thrombin-activated washed rat platelets (or platelet-free supernatants prepared from thrombin-activated platelets) with cultured rat aortic smooth muscle cells induced cholesteryl ester lipid droplet accumulation within the smooth muscle cells. No cholesteryl ester lipid droplets accumulated when smooth muscle cells were incubated with unactivated platelets. Smooth muscle cell lipid droplet accumulation occurred in the absence of serum lipoproteins and was not inhibited by mevinolin, a drug that blocks cholesterol synthesis. These findings suggest that activated platelets may release cholesterol, which can be accumulated by cells and stored as lipid droplets.