乙醇及乙醛对胚胎发育的联合作用

为探讨乙醇及其代谢产物乙醛在胎儿酒精综合征 (FAS)中的作用 ,应用植入后全胚胎培养研究不同浓度的乙醇 ,乙醛组合对 9.5- 1 1 .5d大鼠胚胎发育的联合作用 .结果显示在卵黄囊直径 ,头长 ,体长 ,心脏 ,前脑 ,DNA含量等指标乙醇与乙醛存在交互作用 .进一步分析发现 0 .1 g·L-1乙醇与不同剂量乙醛组合可导致拮抗 ,协同和独立等多种联合作用类型 ,但对头长和脑均表现为独立作用 . 0 .2 g· L-1乙醇与不同剂量乙醛组合除心脏呈协同作用外其余均为乙醇独立作用 .上述结果提示 FAS中的脑发育异常是乙醇独立作用的结果 ,乙醛则引起FAS中的辅助畸形     

乙醇和乙醛对培养大鼠胚胎卵黄囊细胞膜脂质流动性的影响

为探讨酒精对发育中胚胎卵黄囊 ( VYS)膜功能的影响 .孕 9.5d大鼠胚胎于体外分别给予不同剂量的酒精及其代谢产物乙醛 ,培养 48h后 ,分离胚胎 VYS细胞 .以 1 ,6-二苯己三烯 ( DPH)为荧光探针研究二者对 VYS细胞膜脂质流动性的影响 .结果表明 0 .4g· L-1乙醇 ,2 .0 g· L-1乙醛并不影响 VYS细胞膜脂质的荧光偏振度 ( P)和细胞膜流动度 ( LFU) ,随剂量增高可导致 VYS荧光偏振度降低 ,膜脂质流动度增高 ,相同剂量 ( 2 .0 g· L-1)的乙醇 ,乙醛对 VYS细胞作用无显著性差异 .乙醇 ,乙醛均能改变 VYS细胞膜脂质流动性引起 VYS细胞功能紊乱 ,它很可能在诱发胎儿酒精综合征起重要作用 .     

硫酸铝在体外对大鼠胚胎组织谷胱甘肽含量和卵黄囊细胞膜流动性的影响

目的　为探讨铝的发育毒性及机理。方法孕 9.5d大鼠胚胎于体外培养系统中给予不同剂量的硫酸铝 ,培养 4 8h后 ,观察胚胎生长发育和器官形态分化状况 ;应用二硫代双硝基苯甲酸 (DTNB)直接法测定胚胎组织谷胱甘肽 (GSH)含量 ;以 1,6 二苯己三烯为荧光探剂 ,用荧光偏振技术测定卵黄囊细胞膜脂质流动性。结果　当培养液中铝浓度为1.2mg·L- 1时 ,胚胎生长发育和分化明显被抑制 ;3.0mg·L- 1时 ,畸形胚胎发生率明显升高 ,主要有神经管闭合不全 ,脑发育不良和体翻转不全 ;6 .0mg·L- 1时 ,胚胎组织GSH含量和卵黄囊细胞膜脂质流动性显著降低。上述效应均呈现出一定的剂量 效应 (反应 )关系。结论　铝有潜在的致畸性和胚胎毒性 ,胚胎组织GSH含量和卵黄囊细胞膜流动性降低可能在铝致胚胎发育毒性中起重要作用     

乙醇抑制原代大鼠星形细胞神经营养因子的基因表达

为了阐明乙醇中毒对神经系统损害的机理 ,用0 .5- 5.0 g· L-1浓度的乙醇处理原代培养的 CD新生大鼠大脑皮层星形细胞 ,用反转录 PCR和Northern印迹杂交方法同时测定神经营养因子( NGF)的基因表达水平 .结果显示 0 .5g·L-1乙醇在染毒 2 4 h后 ,NGF m RNA水平开始降低 ,5.0 g· L-1在染毒 48或 72 h后 ,NGF m RNA水平与对照组比较降低了 50 % - 58% .结果表明 ,乙醇中毒在体外可降低新生大鼠星形细胞 NGF m RNA的生成 ,这种作用可干扰神经元与胶质细胞间的正常交互作用并可能与胚胎发育期乙醇接触导致的神经病理改变有关 .     

竹节人参皂苷对乙醇致肝细胞L-O2损伤的保护作用

目的考察竹节人参皂苷对乙醇损伤肝细胞L-O2的保护作用,并探讨其作用机制。方法高效液相色谱-蒸发光散射检测法(HPLC-ELSD)分离纯化竹节人参皂苷,MTT法测定细胞存活率,分光光度法测定肝细胞内液丙二醛(MDA)含量、超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)活性。结果经分离纯化鉴定获得了6个竹节人参皂苷单体Rg1,Re,Rf,F3,Rg2和Rd;其中Rg10.16 g·L-1,Re 1.28g.L-1和Rf0.64 g·L-1可促进正常肝细胞L-O2增殖,增殖率分别为22.7%,34.8%和28.5%(P<0.01);Rd0.16 g·L-1和F31.28 g·L-1对正常肝细胞生长表现出明显的抑制作用,抑制率分别为49.7%和43.3%(P<0.01)。Rg10.16 g·L-1,Re 1.28 g·L-1和Rf 0.64 g·L-1对乙醇200 mmol·L-1损伤的肝细胞L-O2具有明显的保护作用,对细胞生长的抑制率由乙醇损伤模型组的50.4%分别降低为23.3%,26.9%和26.6%(P<0.01);Rd 0.16 g·L-1和F31.28 g·L-1则加强乙醇对肝细胞的损伤,抑制率高达83.2%和64.8%(P<0.01)。乙醇损伤模型组肝细胞L-O2乙醇代谢产生MDA含量升高、SOD和GSH-Px降低;Rg10.16 g·L-1,Re 1.28 g·L-1和Rf 0.64 g·L-1可明显改善乙醇损伤导致的肝细胞L-O2内MDA含量升高,增强SOD和GSH-Px活性(P<0.01)。结论人参皂苷Rg1,Re和Rf对乙醇损伤肝细胞L-O2具有明显的保护作用,抗氧化活性可能是其发挥保护作用的机制之一。     

金黄色破囊壶菌发酵生产DHA的研究

研究碳源、谷氨酸钠、盐浓度、培养温度等对金黄色破囊壶菌Thraustochytrium aureum H0细胞生长和DHA产量的影响,测定了发酵过程菌体生长曲线。结果表明,葡萄糖是金黄色破囊壶菌发酵生产DHA的最佳碳源,培养基中葡萄糖、谷氨酸钠最适浓度为30g·L-1和5g·L-1,培养基最适盐浓度为自然海水盐浓度的1/2。金黄色破囊壶菌在优化培养基中发酵4d,菌体量和DHA产量分别达到6.2g·L-1、0.51g·L-1。     

芙朴感冒颗粒对斑马鱼胚胎发育的影响

摘要：目的:研究芙朴感冒颗粒对斑马鱼胚胎发育的影响,为临床安全用药提供相关参考资料。方法:选择6 h的斑马鱼胚胎为实验模型,设置空白对照组和芙朴感冒颗粒低、中、高（10,20,50 g·L-1）浓度组,显微镜下观察并记录斑马鱼胚胎发育情况,检测自主运动次数、心率、畸形率、孵化率、泳速等,以初步分析芙朴感冒颗粒对斑马鱼胚胎发育的影响。结果:芙朴感冒颗粒可致斑马鱼胚胎发育畸形,主要表现为心包囊肿、充血和脊柱弯曲;芙朴感冒颗粒能明显抑制斑马鱼胚胎自主运动和心率;芙朴感冒颗粒低浓度时促进胚胎孵化和斑马鱼胚胎孵育后仔鱼的泳速,高浓度则抑制胚胎孵化和仔鱼泳速。结论:芙朴感冒颗粒可影响斑马鱼胚胎的正常发育。     

甲醛对植入后小鼠胚胎发育毒性的时间-效应关系*

目的:探讨甲醛对植入后小鼠胚胎发育毒性及时间-效应关系。方法:应用体外全胚胎培养模型,观察终浓度为81μmol.L-1的甲醛与昆明种小鼠全胚胎共培养不同时间(分别为0,16,26,48 h)对胚胎生长发育的影响。结果:甲醛对小鼠胚胎的器官形成早期具有明显的影响,呈现明显的时间-效应关系。甲醛暴露16 h则能引起胚胎发育迟缓。当甲醛暴露时间为26和48 h时,胚胎生长发育和形态分化等各项指标与对照组(0 h)比较均呈现出显著差异(P<0.05,P<0.01);胚胎畸形发生率也显著升高,主要表现为神经管闭合不全、体位翻转不全、脑、心脏、腮弓发育异常及视觉系统、前肢芽、后肢芽、体节等畸形。结论:甲醛对小鼠胚胎生长发育和器官形态分化均有明显的抑制作用,表现出明显的时间-效应关系。     

四溴双酚A对斑马鱼胚胎发育毒性和神经毒性研究

目的 研究四溴双酚A (Tetrabromobisphenol A,TBBPA)对斑马鱼胚胎的发育毒性和神经毒性.方法 采用斑马鱼胚胎模型,利用胚胎暴露实验分析剂量效应和畸形表观等发育毒性指标;利用胚胎自主运动、接触反应和仔鱼游泳运动,分析神经毒性指标.结果 TBBPA对斑马鱼胚胎的发育具有中度毒性,在受精后48 h(Hours Post Fertilization,hpf)的半数致死量(Concentration that led t0 50％ mortality,LC50)和半数致畸量(Concentration that led t0 50％ malformations,EC50)分别是7.9和4.7 μmol·L-1,以及在120 hpf的LC50和EC50分别是5.3和1.9 μmol·L-1.发育毒性效应具体表现为(1)在48,60,72,96 hpf均表现为低浓度促进孵化,高浓度抑制孵化;(2)引起胚胎发生尾部弯曲、未吸收卵黄囊、心包囊肿、卵黄囊水肿、游囊关闭等畸形现象;(3)胚胎畸形率和死亡率均具有剂量依赖效应,即与暴露浓度成正比.TBBPA对斑马鱼的神经毒性表现为(1)增加胚胎在19～26 hpf的自主运动频率;(2)降低胚胎在27,36,48 hpf时的接触反应能力;(3)降低胚胎在120 hpf的行为运动速度.结论TBBPA对斑马鱼胚胎具有发育毒性和神经行为毒性.     

阿芬太尼对再灌注心肌功能的保护作用及其机制

目的 :研究阿芬太尼对缺血再灌注心肌功能的保护作用及其作用机制。方法 :采用Langendorff离体大鼠心脏模型 ,以停灌的方式造成全心缺血2 5min ,然后复灌 30min ,药物于缺血前 10min给予并持续到复灌末。观察 5 0 μg·L- 1和 10 0 μg·L- 1阿芬太尼及其与纳洛酮和L NAME合用时对缺血前及再灌注期间左心功能的影响 ,并测定再灌注末时心肌组织ATP和NOS的含量。结果 :(1)在非缺血情况下 ,10 0 μg·L- 1阿芬太尼能使HR减慢 ,对LVEDP、LVDP、±dp/dtmax 和CF无明显影响 ;(2 )5 0 μg·L- 1和 10 0 μg·L- 1阿芬太尼均能明显促进再灌期间左心功能和冠脉流量的恢复 ,且 10 0 μg·L- 1阿芬太尼比 5 0 μg·L- 1阿芬太尼作用更明显 ;(3)2 0 0 μg·L- 1纳洛酮和 10 0 μmol·L- 1L NAME均削弱了阿芬太尼对缺血再灌注心肌功能恢复的有利作用。结论 :阿芬太尼对离体大鼠的心肌功能影响轻微且能促进缺血再灌注后心肌功能的恢复 ,其作用机制可能与阿片受体和内皮细胞释放的一氧化氮有关。     

Comparison of in vitro and in utero ethanol exposure on indices of oxidative stress

Prenatal ethanol exposure produces neural tube defects and growth retardation in experimental animals. Because ethanol's teratogenic effects may involve oxidative stress and effects may differ in vitro and in utero, glutathione, cysteine and ATP were evaluated in gestational day 10 rat conceptuses exposed to ethanol. Cultured embryos exposed to ethanol (1.5 or 3.0 mg/mL) maintained a concentration-dependent decrease in glutathione of 21 or 35%, respectively, at 6 h; visceral yolk sac (VYS) glutathione (GSH) decreased by 22 or 18%, respectively, at 3 h. Maternal ethanol exposure (4.5 g/kg) decreased glutathione by 30% in embryos and VYSs at 3 h, but values rebounded. Cultured embryonic cysteine decreased after 30 min by 42% with both doses and after 6 h by 32 or 38% with 1.5 or 3.0 mg/mL, respectively. Ethanol (1.5 mg/mL) increased VYS cysteine by 35% after 30 min. In utero ethanol exposure decreased embryonic cysteine by 58% at 3 h. Ethanol (1.5 mg/mL) decreased adenosine triphosphate (ATP) by 30-60% in embryos and VYSs at 30 min. After 6 h, embryonic ATP decreased by 41 and 30% with 1.5 and 3.0 mg/mL, respectively, while VYS ATP decreased by 38% with 1.5 mg/mL. In utero ethanol exposure decreased ATP by 31% at 3 h in VYSs. While decreases in GSH and cysteine were evident earlier in utero than in vitro, values returned to control suggesting embryos exposed in utero respond rapidly to chemical-induced oxidative stress due to maternal protective mechanisms. Differences between in vitro and in utero responses to ethanol have important implications for interpretation of in vitro developmental studies.     

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC₅₀) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.     

Spatial distribution of glutathione, glutathione-related and antioxidant enzymes in cultured mouse embryos

The present study was undertaken to evaluate the detoxifying capacity of organogenesis-stage murine concepti cultured in vitro. Investigative attention was particularly focused on the embryonic tissue distribution of cytoprotective pathways. Glutathione (GSH) status, GSH-related and antioxidant enzymes were assayed in the embryo proper (EP), visceral yolk sac (VYS) and ectoplacental cone (EC) of 29.44 ± 1.56 (mean ± SD) somite pairs concepti. All the tissues displayed significant and comparable concentrations of GSH, further supporting this tripeptide as critical in protection against embryotoxicants. The totality of enzymatic activities was detectable in the selected embryonic compartments. In terms of spatial distribution analysis, maximal activities were found in EC (glutathione peroxidase, glutathione reductase, superoxide dismutase and glyoxalase I and II), and VYS (glutathione transferase and catalase). These results indicate: (1) the organogenesis-stage conceptus, in addition to significant amounts of GSH, expresses constitutive activities of GSH-related and antioxidant enzymes; (2) maximal activity levels are detectable in the embryonic sites which, at the developmental stage selected for assay, serve (VYS) or are evolving to serve (EC) embryo/maternal exchange, and thus represent the primary sites of interaction with foreign compounds. Received: 13 March 1997 / Accepted: 18 August 1997     

Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions

Pre-natal alcohol exposure induces delays in fine and gross motor skills, and deficiencies in reflex development via mechanisms that remain to be elucidated. The purpose of the present study was to investigate the effect of embryonic ethanol exposure (16-hour exposure window with1.5%, 2% or 2.5% EtOH) on synaptic properties at the neuromuscular junction (NMJ) in 3 day post fertilization (dpf) zebrafish larvae. Immunohistochemical studies show that exposure of embryos to 2.5% ethanol for 16 h results in motor neuron axons that display abnormal branching patterns. Co-labelling embryos with pre-synaptic markers such as SV-2 or 3A10, and the post-synaptic marker, α-bungarotoxin, which irreversibly binds to nicotinic acetylcholine receptors (nAChRs), indicates that pre- and post-synaptic sites are properly aligned even when motor neuron axons display abnormal morphology. Miniature endplate currents (mEPCs) recorded from muscle fibers revealed the presence of two types of mEPCs that we dubbed fast and slow. Ethanol treated fish experienced significant changes in the frequencies of fast and slow mEPCs, and an increase in the rise time of slow mEPCs recorded from red muscle fibers. Additionally, embryonic exposure to ethanol resulted in a significant increase in the decay time of fast mEPCs recorded from white fibers. Mean mEPC amplitude was unaffected by ethanol treatment. Together, these results indicate that zebrafish embryos exposed to ethanol may experience altered synaptic properties at the NMJ.     

Uptake and elimination of ethanol by young zebrafish embryos

Among animal models being explored to understand ethanol-induced teratogenesis, the zebrafish (Danio rerio) is attracting attention because its embryonic development is well characterized and readily visualized. Despite the potential of the zebrafish embryo in research on developmental anomalies produced by ethanol exposure, little is known about the relationship between embryonic ethanol content and the nature/severity of ethanol-mediated deficits. Here, using gas chromatography and radiometry of labeled ethanol carbon, we examine accumulation and clearance of ethanol by dechorionated zebrafish embryos during blastulation/gastrulation. Our data indicate that: (a) rates of uptake and loss of ethanol are directly proportional to the extra-/intra-embryonic ethanol concentration gradient and (b) ethanol in the water fraction of embryos reaches near equimolarity with ethanol in the exposure medium. It appears that, within a wide range of exposure concentrations, embryonic ethanol content can be predicted accurately according to exposure time. Furthermore, it appears that embryonic ethanol can be adjusted rapidly to and maintained at a targeted concentration.     

Effects of embryonic exposure to ethanol on zebrafish visual function

Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS).     

用全胚胎培养方法评价重铬酸钾对大鼠和小鼠胚胎的发育毒性

采用植入后全胚胎培养方法研究重铬酸钾对大,小鼠胚胎的发育毒性. 结果表明,重铬酸钾在1.0和2.5 mg·L^-1以上时分别可致大,小鼠胚胎体长,头长,卵黄囊直径及胚胎干重等各项指标明显低于对照组,同时观察到大,小鼠胚胎脑,神经管和体屈等发育迟缓,组织光镜可见胚胎神经管上皮变薄,细胞排列不规则. 以上结果均呈明显的剂量 反应关系,表明重铬酸钾体外对大,小鼠胚胎具有明显的发育毒性,且大鼠比小鼠敏感.