In vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cells, the gill cell line of flounder Paralichthy olivaceus

The in vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cell line, derived from the gill of flounder Paralichthys olizaceus, was tested by the three widely used endpoint bioassays-neutral red （NR） assay, tetrazolium （MTT） assay and cell protein assay. It was found that acetamiprid was increasingly toxic to FG cells at concentrations of 1 μg/cm^3 or above, and the inhibitory concentration 50% values for NR, MTF, and cell protein assays were 38.38, 36.27 and 32.03 μg/cm^3, respectively. This appeared to be the first report on the in vitro cytotoxicity of acetamiprid to non-mammalian vertebrate cells. Ultrastructural examination revealed that for the cells exposed to 60 μg/cm^3 acetamiprid for 48 h, their mitochondria were severely damaged with the cristae swelled up or disrupted, while their nuclei and rough endoplasmic reticlum （RER） appeared to be still normal. This suggests that mitochondria are possibly the primary target of acetamiprid.     

金钱鱼鳃细胞系的建立及其生长特性的初步研究

鳃是鱼类渗透压调节的主要器官, 鳃细胞的培养技术的建立可以为鱼类渗透压调节机理研究提供重要的实验手段和研究方法。金钱鱼(Scatophagus argus)作为一种广盐性鱼类, 是研究渗透压调节机制的理想动物模型。本研究采用胰蛋白酶消化法进行了金钱鱼鳃细胞的体外培养, 确定该类细胞原代和传代培养的最优条件, 分析了其生长特性。结果表明, 金钱鱼鳃细胞系在28℃, 含有20%胎牛血清(fetal bovine serum, FBS)的L-15培养基中生长最佳, 2~4d即可传代, 传代后可稳定培养, 命名为SG。在传代培养过程中, 对其增殖情况进行分析, 发现SG细胞群体倍增时间为40.8h。正常传代的SG细胞冻存、复苏后, 经台盼蓝染色测得细胞存活率约为87%。鳃细胞直接同水体环境接触, 具有高效的渗透压调节能力。环境盐度变化时, 鳃细胞启动渗透压胁迫应答机制, 维持内环境稳态。本研究对SG细胞进行渗透压刺激后, 检测其增殖情况并观察形态变化。分析表明, SG细胞在分别为150和600mOsmol·kg ^-1的低渗和高渗胁迫后均可增殖, 且低渗胁迫后的增殖速度是高渗胁迫的1.5倍。渗透压胁迫后SG细胞的形态观察结果显示, 低渗培养基胁迫后细胞体积发生膨胀而高渗条件下细胞体积发生皱缩。由此推断SG细胞具有较强渗透压耐受性, 且低渗耐受能力强于高渗。SG细胞系的建立为金钱鱼渗透压应答机制和相关基因功能的研究提供了基础实验材料。     

氯化锌对松江鲈肾细胞系的毒性效应

本文研究了不同浓度的氯化锌对55~65代松江鲈(Trachidermus fasciatus)肾细胞系的毒性效应。采用MTT法测得氯化锌24 h半致死浓度为308.41μmol/L。酶活力测定的结果显示:超氧化物歧化酶(SOD)和谷胱甘肽-S-转移酶(GST)的活性在氯化锌浓度为0~250μmol/L时,随浓度的升高逐渐升高;在250μmol/L浓度时达到最大值,而后随着氯化锌浓度的升高逐渐降低。谷胱甘肽过氧化物酶(GSH-Px)的活性在氯化锌浓度为125μmol/L浓度时达到最大值,而后随着氯化锌浓度的升高逐渐降低。微核实验结果是微核率随氯化锌浓度增加而增加,最高达12.33‰。彗星实验发现在半致死浓度条件下松江鲈肾细胞拖尾率为48.00%、迁移长度15.03μm±2.51μm,与对照组差异显著(P0.05)。以上结果表明:氯化锌会引起肾细胞氧化酶活性的改变以及细胞核DNA损伤,本研究认为松江鲈肾细胞系可以作为Zn2+污染的监测平台。     

中国明对虾囊胚和原肠胚细胞的分离和培养

尝试了多种方法分离中国明对虾(Fenneropenaeus chinensis)囊胚细胞和原肠胚细胞并进行了细胞培养.结果表明,解剖法适用于囊胚细胞,自行设计的压片法适用于原肠胚细胞.在培养起始阶段,通过降低血清浓度和缩小培养体系可使中国明对虾囊胚细胞和原肠胚细胞迅速贴壁并铺展,囊胚细胞铺展后有明显的生长晕,中后期原肠胚细胞较易达到半汇聚状态.值得注意的是,中国明对虾囊胚细胞在不同培养液中培养可发生形态上的分化.本实验方法有望用于对虾细胞分化机理和对虾细胞建系的研究.     

Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis)

A new cell line,CSEC,has been successfully established from embryos at gastrula stage of a cultured marine fish,half smooth tongue sole(Cynoglossus semilaevis).CSEC cells grow actively and stably more than 50 passages for over 200 d in DMEM medium supplemented with 15% FBS(fetal bovine serum),2.5 ng/cm 3 bFGF(basic fibroblast growth factor),1 ng/cm 3 LIF(leukemia inhibitory factor) and 50 mmol/dm 3 2-ME(2-mecaptoethanol).The cells grew well in the temperature range of 24-30 ℃ and the optimal growth temperature was 24℃.FBS and bFGF concentrations are the two key components for CSEC cells proliferation.Chromosome analysis reveals that CSEC cells have a normal diploid karyotype with 2n=42t.The significant fluorescent signals were observed in CSEC after transfection with the GFP reporter gene,suggesting that the CSEC cell line can be used as a useful tool for transgenic and genetic manipulation studies.CSEC cells showed the cytopathic effect(CPE) after infection with lymphosystis disease virus(LCDV) in 2 d.Moreover,the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy.It suggests that CSEC could be potentially used for the study of aquatic virus.