Sal B对肝癌细胞株HepG2的促凋亡作用

目的揭示丹酚酸B(Salvianolic Acid B,Sal B)对肝癌细胞株HepG2的杀伤作用。方法用不同浓度的丹酚酸B处理HepG2细胞,37℃培养24h。用RT-PCR检测促凋亡基因Bax的转录水平,并用流式细胞术检测细胞凋亡的水平。结果①100μmol/L、50μmol/L、25μmol/L等浓度的Sal B处理都能使HepG2细胞促凋亡基因bax的转录水平升高,其中100μmol/L处理组最为明显。②不同浓度的Sal B处理都能使HepG2细胞发生凋亡,其中100μmol/L处理组最为明显。结论 Sal B有促进HepG2细胞凋亡的作用。     

铜、锌、镉对唐鱼胚胎及初孵仔鱼的急性毒性及安全浓度评价

采用静水法生物测试研究铜（Cu2＋）、锌（Zn2＋）和镉（Cd2＋）对唐鱼（Tanichthys albonubes）胚胎和初孵仔鱼的急性毒性及安全浓度评价。结果显示,Cu2＋对唐鱼胚胎12、24h LC50分别为2.4092mg/L和0.4039mg/L;Zn2＋和Cd2＋对唐鱼胚胎24h LC50分别为372.9mg/L和50.0mg/L。Cu2＋对唐鱼初孵仔鱼12、24和48h LC50都是0.3228mg/L,其安全浓度为0.0986mg/L;Zn2＋对唐鱼初孵仔鱼24、48h LC50分别为72.44mg/L和25.17mg/L,其安全浓度为0.9116mg/L;Cd2＋对唐鱼初孵仔鱼24、48h LC50分别为36.5mg/L和20.59mg/L,其安全浓度为1.9654mg/L。结果表明,重金属元素对唐鱼胚胎和初孵仔鱼毒性依次为Cu2＋〉Cd2＋〉Zn2＋。     

纳米ZnO对斑马鱼肝脏生理生化指标的影响

研究了纳米ZnO对斑马鱼肝脏生理生化指标的影响。当斑马鱼暴露在ZnO纳米颗粒浓度为2.8 mg/L、5.6mg/L、11.2 mg/L、22.4 mg/L和44.8 mg/L时,分别测定了6 h、12 h、24 h、48 h和72 h时斑马鱼肝组织中的GSH、MDA、Na＋K＋-ATPase的含量和24 h ROS的变化,结果显示：与对照组相比,处理组中斑马鱼肝组织中GSH的含量显著减少（P〈0.05）,MDA含量随处理浓度的升高却显著增加（P〈0.05）,Na＋K＋-ATPase活性随暴露时间先显著降低后显著升高（P〈0.05）,24 h ROS含量高于对照组（P〈0.05）,说明斑马鱼肝组织的生理活动受到ZnO纳米颗粒的影响,且有时间和浓度依赖性。     

富马酸二甲酯对斑马鱼胚胎早期发育的影响

为研究富马酸二甲酯对斑马鱼(Danio rerio)胚胎早期发育的影响,选取不同发育阶段的斑马鱼胚胎,用富马酸二甲酯进行染毒处理,观察胚胎形态发育的异常,计算其对不同发育时期胚胎的24 h、48 h半数致死浓度(LC50)和胚胎72 h孵化率,并考察富马酸二甲酯对胚胎血管发育的影响。结果表明,富马酸二甲酯影响斑马鱼胚胎的早期发育,呈剂量依赖性特点,并与开始处理的时间点有关。富马酸二甲酯引起2 hpf(受精后2 h,2 hours post-fertilization)、10 hpf、24 hpf斑马鱼胚胎死亡的24 h LC50值分别为:13.33μmol/L、17.98μmol/L、32.50μmol/L,48 h LC50值分别为:13.31μmol/L、16.35μmol/L、22.50μmol/L;长期低浓度富马酸二甲酯(≥6μmol/L)作用引起胚胎72 h孵化率下降。27.5μmol/L富马酸二甲酯作用后会显著降低胚胎血管内皮生长因子受体2(VEGFR2)的表达水平。     

铅和邻苯二甲酸二丁酯联合暴露对斑马鱼胚胎的影响

为了研究铅(Pb)和邻苯二甲酸二丁酯(DBP)单独及联合暴露对斑马鱼胚胎神经发育和细胞凋亡相关基因表达的影响,考察两者对斑马鱼胚胎神经系统的毒性作用。将720个斑马鱼胚胎按3×3析因设计随机分为9组,以Pb(0、0.01 mg/L和1 mg/L)和DBP(0、0.005 mg/L和0.5 mg/L)单独及联合对斑马鱼胚胎暴露120 h。观察、记录胚胎的孵化、死亡情况,采用荧光定量PCR测定斑马鱼胚胎神经发育相关基因(NR1A、NR2A、NR2D)和神经细胞凋亡相关基因(bcl-2、c-fos)的表达变化情况。与空白对照组相比,各暴露组均可致斑马鱼胚胎孵化率降低、死亡率增高(P0.05);联合暴露组NR1A、NR2A、NR2D、c-fos基因表达水平显著升高,bcl-2基因表达水平显著下降(P0.05)。Pb和DBP均可影响斑马鱼胚胎的发育,且具有一定的神经毒性,而二者联合暴露毒性复杂,对斑马鱼胚胎孵化率和死亡率的影响有交互作用,对神经发育和细胞凋亡相关基因无交互作用,其机制有待进一步研究。     

1-苯基-2-硫脲对斑马鱼胚胎发育与黑色素生成的影响

目的1-苯基-2-硫脲（PTU）可抑制斑马鱼胚胎黑色素的产生,保持斑马鱼透明,便于形态观察和信号检测。本文研究了PTU对斑马鱼胚胎发育的影响和抑制斑马鱼胚胎黑色素生成,保持斑马鱼透明性的最佳浓度。方法用不同浓度PTU处理23hpf（受精后,hourspostfertilization,hpf）斑马鱼胚胎,作用57h后观察80hpf斑马鱼的形态学、生理学改变,计算死亡率和孵化率,测量心率和静脉窦-动脉球之间的距离。结果浓度为0．197mmo]／L、0．296mmol／LPTU可以有效抑制黑色素生成,保持斑马鱼整体透明,对斑马鱼心血管系统结构和生理功能无影响,且不影响斑马鱼正常孵化过程。随着PTU浓度的增加,斑马鱼死亡率增加,孵化率下降,出现心包水肿,心脏畸形等改变,心率下降,静脉窦-动脉球之间的距离增大。结论浓度不高于0．296mmol／L的PTU溶液能有效抑制斑马鱼黑色素生成,对斑马鱼心血管毒性研究无影响。     

乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2表达和caspase-3活性的影响

目的：探讨乳酸堆积和二氯乙酸钠（OCA）对肝癌细胞（HepG2）凋亡和bax、bcl-2表达及caspase-3活性的影响。方法：通过体外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0mmol／L、1．0mmol／L、2．0mmol／L、4．0mmol／L、8．0mmol／L的乳酸培养液以及在不同浓度乳酸组中加入终浓度为10^-3mmol／LDCA培养液与HepG2共同培养,其中以0mmol／L乳酸组为对照组。采用MTT法检测乳酸对HepG2的抑制率,流式细胞仪检测乳酸和DCA对HepG2的凋亡百分率,用Real-time PCR法测定bax及bcl-2mRNA的表达,用免疫荧光法检测caspase-3的活性。结果：乳酸对HepG2的IC50值为13．6mol／L,与对照组比较,随着乳酸浓度的增加,HepG2凋亡率增加,baxmRNA表达升高,bcl-2mRNA的表达降低,caspase-3活性增加,其中1．0mmol／L乳酸组与对照组比较（P〉0．05）,2．0mmol／L,4．0mmol／L和8．0mmol／L乳酸组与对照组比较差异有统计学意义（P〈0．05）。加入DCA后．HepG2凋亡减少,2．0mmol／L乳酸＋DCA组、4．0mmol／L乳酸＋DCA组、8．0mmol／L乳酸＋DCA组与同浓度的乳酸组比较,baxmRNA表达减少（P〈0．05）,bcl-2mRNA表达增加（P〈0．05）,caspase-3活性减低（P〈0．05）。结论：乳酸可诱导HepG2凋亡,且随着乳酸浓度的增高,HepG2的凋亡率增加,其机制可能是通过对bcl-2及baxmRNA表达的改变以及激活caspase-3活性而实现,DCA可以降低HepG2凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。     

迷迭香酸对硫酸铜致斑马鱼胚胎毒性的抑制作用

本文探讨硫酸铜(CuSO_4)对斑马鱼(Danio rerio)胚胎发育的毒性效应,使用迷迭香酸(RA)抑制CuSO_4对斑马鱼胚胎发育的毒性并探讨其作用机制。收集受精后1 h(1 hpf)的斑马鱼胚胎暴露于不同浓度的CuSO_4溶液,或含有不同浓度迷迭香酸的CuSO_4溶液,对照组培养在E3培养液中,观察胚胎死亡、孵化及畸形情况,计算胚胎死亡率、孵化率和畸形率;以活性氧(ROS)荧光探针DCFH-DA染色法检测迷迭香酸保护下胚胎的活性氧水平。对实验数据进行方差分析。结果显示:(1)CuSO_4浓度超过一定量时能诱导斑马鱼胚胎死亡和畸形,胚胎孵化率也降低。CuSO_4对96 hpf斑马鱼胚胎的半致死浓度(LC50)为7.7μmol/L,半致畸浓度(EC50)为1.9μmol/L。(2)在96 hpf,迷迭香酸与8μmol/L CuSO_4共同处理组斑马鱼胚胎的死亡率明显降低,孵化率升高。迷迭香酸与1.6μmol/LCuSO_4共同处理组斑马鱼胚胎的畸形率降低。(3)CuSO_4单独处理组的活性氧含量明显高于迷迭香酸与CuSO_4共同处理组和对照组。结果表明,CuSO_4暴露对斑马鱼胚胎发育的毒性效应可能与活性氧升高导致的氧化应激相关;迷迭香酸抑制CuSO_4对斑马鱼胚胎发育的毒性作用,可能与减少活性氧生成有关。     

餐厨垃圾高温菌种对斑马鱼急性毒性与安全评价

目的:研究餐厨垃圾高温菌种对斑马鱼的毒性,为其安全使用提供依据。方法:以具有代表性的水生动物斑马鱼为试材,采用静态法测定餐厨垃圾高温菌种对斑马鱼急性毒性并对其进行安全评价。结果:鱼的半数致死浓度LC50值24、48和96h分别为3400mg/L、3000mg/L和2500mg/L。结论:餐厨垃圾高温菌种对斑马鱼有一定毒性,该研究为餐厨垃圾高温菌种的安全合理使用提供了科学依据。     

外源性视黄酸对斑马鱼心血管系统发育的影响

目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。     

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure

The acute zebra fish embryo test (Danio rerio Hamilton-Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC₅₀ below 1.2 µmol compared with an EC₅₀ of 91 µmol. The dose-response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.     

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure

Abstract

The acute zebra fish embryo test (Danio rerio Hamilton–Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC₅₀ below 1.2?µmol compared with an EC₅₀ of 91?µmol. The dose–response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.     

姜黄素类似物EF24诱导A549细胞自噬及凋亡关系的研究

从细胞自噬及凋亡关系角度探讨姜黄素类似物EF24对人肺腺癌细胞（A549）的杀伤机理。选用不同浓度的EF24对体外培养的A549处理,采用MTT方法检查细胞存活率,吖啶橙染色观察细胞形态,蛋白质免疫印迹（Western blot）方法检测与细胞自噬及凋亡相关蛋白的表达及对AMPK-mTOR-S6K信号通路的影响。结果显示,EF24作用24 h的IC50为8.5μmol/L,对A549细胞生长抑制作用优于姜黄素,而接近顺铂。自噬及凋亡蛋白检测显示,在4μmol/L、8μmol/L时A549细胞以自噬为主,在16μmol/L时以凋亡为主;加入100 nmol/L自噬抑制剂渥曼青霉素（wortmannin）后,细胞存活率同比升高。同时还发现,随着EF24浓度的增加,细胞内AMPK-Thr172磷酸化水平上升,mTOR-Ser2481、S6K-Thr389磷酸化水平的下调。由此可见,EF24可通过AMPK的激活下调mTOR-S6K途径抑制细胞生长,在EF24浓度4~8μmol/L范围内,自噬对凋亡起到促进作用。     

无瓣海桑果叶及其浸提液的生物急性毒性研究

为了探讨无瓣海桑的生物毒性作用,采用哺乳类动物大鼠,国际规定的标准毒性实验用鱼斑马鱼以及红树林内主要鱼种弹涂鱼为受体,进行了大鼠急性经口毒性固定剂量毒性反应观察,斑马鱼和弹涂鱼的改进寇氏法毒性实验以及半致死量浓度(LC₅₀)和95%可信限计算,结果表明:在5000 mg/kg体重下,无瓣海桑的果实和叶子浸提液都未对大鼠产生任何毒性表现,14 d的死亡率为0,半致死量(LD₅₀)大于5000 mg/kg体重,属于无毒物质;对鱼类毒性,相同鱼种下,果实水浸提液毒性强于叶子水浸提液,相同水浸提液下,弹涂鱼对毒性敏感度超过斑马鱼,半致死浓度24 hLC₅₀>4 8 hLC₅₀>72 hLC₅₀>96 hLC₅₀>1000 mg·L^-1,属于低毒物质.根据以上初步得出无瓣海桑不会对周围生物造成毒性危害.     

PP2A-A/在金鱼及斑马鱼发育中的分化表达模式

以金鱼和斑马鱼为研究对象,运用RT-PCR和Western Blot技术分析蛋白磷酸酶2A(PP2A)结构亚基A(PP2A-A/)在金鱼、斑马鱼成体9种组织和12个发育时期胚胎中mRNA和蛋白水平的表达情况,得到其分化表达模式为:(1)在mRNA水平上,PP2A-A/在金鱼、斑马鱼9种组织中具有较强表达;种属差异性和组织差异性均较大;结构亚基A的两亚型A和A的表达存在差异。(2)在蛋白水平上,PP2A-A/在金鱼、斑马鱼9种组织中均有表达;种属差异性不大但出现明显的组织差异性。(3)PP2A-A/mRNA在金鱼和斑马鱼卵裂期到囊胚期胚胎中大量存在,PP2A-AmRNA在金鱼眼色素期量剧增推测其对金鱼眼色素的形成至关重要。(4)PP2A-A/基因在金鱼、斑马鱼12个发育时期胚胎中均有较高水平的蛋白存在,提示其为维持胚胎的正常发育发挥重要作用。       

Early differentiation in zebra fish blastula: Role of yolk syncytial layer

Summary The blastomeres of the zebra fish embryo can be classified into two types-cells stained densely (D) or lightly (L) with a mixture of toluidine and methylene (T-M) blue. The dense staining of D cells is largely due to the high density of mitochondria, rough endoplasmic reticulum and polyribosomes. The presence of partially dense stained cells during early blastula stage shows that L cells are transformed into D cells. That the yolk syncytial layer (YSL) plays some role in this transformation is suggested by the proximity of these cells to the YSL and by their distinct spatial orientation with densely stained cytoplasmic regions always facing towards the interior of the embryo.     

Spatio-temporal distribution of gap junctions in zebra fish embryo

Summary The distribution of gap junctions in zebra fish embryo is a dynamic process. They appear during cleavage stages and are uniformly distributed between deep cells in early blastula. By mid-blastula stage their density increases all over the embryo. At this stage lightly stained L cells and densely stained D cells (Dasgupta and Singh 1981; Wilhelm Roux's Arch Dev Biol 190:358) develop some differences in their surface properties due to which the frequencies of gap junctions between homologous (L-L and D-D) cells are two- to threefold higher than between heterologous (L-D) cells. Before the initiation of epipoly the gap junction occurrence increases in the envelopinglayer (EVL) and then decreases. The possible functional significance of the organization of these junctions is discussed.