草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究

以草鱼呼肠孤病毒(GCRV)感染的草鱼肾细胞系(CIK)为模型,进行了草鱼呼肠孤病毒在细胞内的形态发生的研究.当病毒以感染复数为5～10PFU/CELL感染CIK细胞时,在病毒感染细胞4h以内的切片中,可观察到脱去部分外层衣壳的不完整病毒颗粒.感染细胞8h,可观察到浆胞内病毒发生基质,其内含有大量的直径约50nm的亚病毒颗粒,无外层蛋白结构.感染12～16h后,这些亚病毒颗粒装配上外层蛋白结构,形成直径为72nm左右的成熟的病毒粒子.病毒感染细胞8h后,开始出现典型的病毒包含体,16～20h小时病毒包含体裂解,继而释放出有感染性的子代病毒颗粒.该结果有助于对GCRV致病机理的了解.     

MTT法评价医用骨科材料的细胞毒性

目的:评价三种常用医用骨科材料的细胞毒性。方法:通过制备表面阳极氧化钛合金(Ti-6Al-4V)材料、聚醚醚酮(PEEK)材料和β-磷酸三钙(β-TCP)材料的浸提液与L929细胞接触,进行MTT试验。结果:所有样品浸提液的细胞相对增殖率(RGR)均≥80%,细胞毒性反应分级为0至1级。结论:这三类材料的0.2g/ml浸提液均显示无明显的细胞毒性。     

MTT方法的优化

目的：对MTT法检测悬浮细胞增殖活性的实验条件进行筛选。方法：以K562细胞为实验对象,分别测定不同MTT用量、细胞浓度、MTT溶剂种类及作用时间等实验条件下的OD570值。结果：检测K562细胞的增殖活性时,细胞浓度应选取0．8×108～0．2×108/L,MTT加入量不应超过20μL/孔,若不考虑时间成本,应以三联溶液作为甲硂溶剂,反应12 h后检测,所获结果精密度最高;若需快速获得结果,也可选择DMSO作为甲硂溶剂,反应10 min后检测。结论：建立了优化的MTT法检测悬浮细胞增殖活性。     

自体DC、CIK细胞在急性髓细胞白血病治疗中的研究进展

树突状细胞(DC)是初级免疫应答的激发者,是最有活力的抗原递呈细胞(APC),可以有效地抑制白血病细胞逃逸。未成熟DC细胞从细胞外捕获各种抗原信息,成熟DC细胞传递各种抗原信息给宿主淋巴结的T细胞,激活抗原相关的主要组织相容性复合体(MHC)限制性特异性免疫应答,另外,亦可通过影响B细胞的增殖,不同程度的活化体液免疫应答。细胞因子诱导的杀伤细胞(CIK)是一组具有细胞毒作用的异质细胞群,是较LAK细胞溶瘤活性更强的一种免疫活性细胞,对包括白血病在内的多种恶性肿瘤具有抗肿瘤效应,具有非MHC限制性的杀瘤特点。二者联合培养及应用又增强了各自的活性。DC细胞与CIK细胞对于白血病的疗效不仅在实验室得以证实,而且已经逐步应用于临床,其在清除微小残留病以及预防造血干细胞移植后复发中取得了良好的效果。随着细胞制备技术的完善和研究的进一步深入,自体DC、CIK细胞治疗急性髓细胞白血病逐渐获得众多专家的认可。中国卫生部已经把自体免疫细胞治疗技术做为第三类医疗技术应用于临床,批准号200984。本文就目前DC、CIK、DC-CIK细胞免疫疗法在急性髓细胞白血病中的应用进展加以综述。     

MTT法测定眼镜蛇毒细胞毒素的抗癌活性

目的测定眼镜蛇毒细胞毒素（Ｃｏｂｒａｖｅｎｏｍｃｙｔｏｔｏｘｉｎ,ＣＶ－ＣＴＸ）对体外培养的人癌细胞的杀伤作用。方法溴化二苯四偶氮盐（ＭＴＴ）比色法。结果ＣＶ－ＣＴＸ对ＳＧＣ－７９０１,Ｂｅｌ－７４０２,Ｋ５６２和Ｕ９３７４种人癌细胞有很强的杀伤作用,量效关系明显,ＩＣ５０分别为４．１０,２．０８,０．２９和０．１７μｇ／ｍｌ。结论ＭＴＴ法检测ＣＶ－ＣＴＸ的抗癌活性,操作简便、快速和准确。     

两种体外细胞毒性检测方法的比较研究

目的比较两种常用的细胞毒性检测方法在医疗器械生物学评价中的相关性。方法分别采用MTT比色法和细胞增殖度法,在37℃条件下,将五种医疗器械／生物材料的浸提液分别与小鼠成纤维细胞（L-929）接触2天和2,4,7天,比较材料对细胞的毒性影响。结果5种不同的材料浸提液分别表现出不同程度的细胞毒性反应（0～2级）。将MTT比色法与细胞增殖度法（2天）的实验数据进行相关性分析,显示两者之间具有良好的相关性（R=0．977）。结论MTT比色法由于其检测所需的细胞量相对较少,试验步骤相对简便、检测周期短,因此具有一定的优越性,是个值得推荐的细胞毒性检测方法。     

铅、镉、铬单一和复合污染对青菜种子萌发的生物学效应

重金属污染是当今世界上倍受重视的一类公害。重金属以汞毒性最大 ,镉次之 ,铅、铬、砷也有相当毒性 ,有人称之为“五毒”。冶炼和采矿是向环境中释放重金属的最重要的污染源。此外 ,不少工业部门也通过三废向环境中排放重金属。农作物受到重金属污染后 ,不仅严重影响其产量和质量 ,更为严重的是重金属经食物链进入人体 ,在人体内富集 ,危害人类健康。近几年 ,国外利用陆生植物进行毒性试验已有所增加 ,其中包括植物种子发芽毒性试验[1] 。植物种子发芽毒性试验较其它毒性试验方法有两大优点 :一是方法简单 ,持续时间短 ;二是这种方法对毒物…     

睡美人转座子在草鱼CIK细胞中介导的高效基因整合

为研究睡美人(Sleeping Beauty, SB)转座子系统在草鱼(Ctenopharyngodon idellus)肾脏细胞(CIK)中介导的整合特性, 构建了SB转座子和转座酶在两个质粒的二元反式(trans)转座子系统, 以及转座子和转座酶元件在同一个质粒的一元顺式(cis)转座子系统; 通过转染CIK细胞, 用荧光显微镜、流式细胞仪和荧光定量PCR分析了转染2d后及嘌呤霉素筛选4周后的细胞, 测定DsRed转染效率和整合效率, 并结合高效热不对称交互式PCR扩增获得SB转座子整合位点的序列。结果表明, SB二元转座子系统的整合效率远高于一元系统; SB转座子与转座酶比例为1﹕2时, 外源基因DsRed的整合效率最高; SB转座子偏向于插入草鱼基因组TA序列处。研究表明优化SB转座子和转座酶的比例能提高外源基因在草鱼细胞中的整合效率并快速获得突变细胞, 同时为在鱼类细胞中采用SB转座子建立突变体文库提供理论基础。     

MTT法用于药物对L2细胞毒性的实验研究

目的：评价噻唑蓝（MTT）法检测药物对细胞的毒性作用的可靠性。方法：大鼠肺泡上皮L2细胞以叔丁基对苯二酚（TBHQ）10．100μM,BsO以1-10mM分别处理,用MTT法检测细胞活性、JC-1（5,5’,6,6’-四氯．1,1’,3,3’-四乙基苯并咪唑羰花青碘化物）荧光染料法检测细胞线粒体电位改变、台盼蓝排斥实验检测细胞死亡率,分析各指标的情况。结果：在处理剂量范围,MTT法检测到的光密度（OD）值未能达到一般判断的半数抑制浓度（ic50）水平,最高抑制率仅达到30％左右;台盼蓝排斥试验检测数据表明TBHQ的LC50值为50μM,丁硫氨酸亚砜胺（BSO）为5mM;利用JC-1荧光染料判断的半数凋亡剂量分别为50μM和7mM。结论：MTT法作为最常采用的细胞生长抑制检测手段,但在某些特定实验中可能不能客观地反映细胞的活性,建议多种方法结合进行评价。     

草鱼肾细胞电转条件的优化及草鱼呼肠孤病毒NS26蛋白的瞬时表达

本研究先以pEGFP-N1载体质粒中的绿色荧光蛋白基因为报道基因进行电转实验,优化电压、脉冲时间、质粒添加量和电击次数等电转条件,确立最佳条件。实验表明,细胞密度为1.5×107/mL时,取200μl在0.2cm电击杯中进行电击转染,电压为200V,脉冲时间为45ms,添加质粒30μg,电击1次时,转染效果最佳。提取草鱼呼肠孤病毒基因组总RNA,以其逆转录的cDNA作为扩增模板,设计特异引物扩增出GCRV非结构蛋白NS26的基因片段,重组到pEGFP-N1载体上得到重组质粒pEGFP-NS26,将其导入CIK细胞中用电转法高效表达了EGFPNS26融合蛋白。本研究为进一步研究NS26的功能奠定了基础。     

Involvement of apoptosis in malathion-induced cytotoxicity in a grass carp (Ctenopharyngodon idellus) cell line

We investigated the role of apoptosis in malathion-induced cytotoxicity in the grass carp (Ctenopharyngodon idellus) cell line ZC-7901. Fish cells were treated with different concentrations of malathion (0.62-95 mg/L), and the IC(50) ranged from 37.94+/-1.93 mg/L for 12 h to 3.04+/-0.27 mg/L for 72 h by the MTT assay. Apoptosis was detected by confocal laser scanning microscopy, transmission electron microscopy, TUNEL reaction, DNA laddering and a flow cytometric PI staining assay. The results demonstrated that apoptosis was involved in the cytotoxic effect of malathion, and that malathion-induced apoptosis occurred in a dose- and time-dependent manner. In addition, the induction of apoptosis by malathion was accompanied by mitochondrial membrane potential (DeltaPsi(m)) disruption, intracellular Ca(2+) elevation, generation of reactive oxygen species (ROS) and ATP depletion. Our investigation suggested that malathion exerts its cytotoxic effects by the induction of apoptosis via a direct effect on the mitochondria.     

青菜幼苗体内几种保护酶的活性对Pb、Cd、Cr胁迫的反应研究

1 引言自从McCord在1969年第一次从牛红血细胞中发现超氧化物歧化酶(SOD)并且证明其功能是清除超氧化物以后,研究者们在植物细胞中也发现了这种酶,并且证明具有同样的功能.后来又发现植物细胞通过多种途径产生活性氧自由基,同时细胞也存在清除这些自由基的多种途径[5],两者形成平衡体系.但是,许多逆境因子如寒冷、干旱、干燥、水淹、重金属Al等都能影响植物体内活性氧代谢系统的平衡,产生大量的氧自由基,它们能够启动膜脂过氧化或膜脂脱脂作     

Assessing cytotoxicity of boron nitride nanotubes: Interference with the MTT assay

Thanks to a non-covalent wrapping with glycol-chitosan, highly biocompatible and highly concentrated dispersions of boron nitride nanotubes were obtained and tested on human neuroblastoma cells. A systematic investigation of the cytotoxicity of these nanovectors with several complementary qualitative and quantitative assays allowed a strong interference with the MTT metabolic assay to be highlighted, similar to a phenomenon already observed for carbon nanotubes, that would wrongly suggest toxicity of boron nitride nanotubes. These results confirm the high complexity of these new nanomaterials, and the needing of extensive investigations on their exciting potential applications in the biomedical field.     

Immunization with recombinant baculovirus expressing the VP6 protein of grass carp reovirus induces immunity in grass carp

Grass carp reovirus（GCRV）,a member of the genus Aquareovirus in the family Reoviridae（Attoui H,et al,2002）,is one of the most virulent aquareoviruses iden-tified so far,leading to high fatality rates（up to 85%）of fingerlings and yearlings of grass carp（Fang Q,et al,2007）.No commercial vaccines against GCRV infection have been developed as yet.     

草鱼和鲢鱼肌球蛋白Ca2+-ATPase热力学活化参数的季节变化

肌球蛋白构成了鱼肌肉肌原纤维蛋白的50%以上,它的性质主要决定了肌原纤维蛋白的性质,进而影响了鱼肌肉蛋白质的加工适性.本文对来自春季(4月份)、夏季(8月份)、秋季(11月份)和冬季(1月份)的草鱼(Ctenopharyngodon idellus Valenciennes)和鲢鱼(Hypophthalmichthys molitrix Valenciennes)骨骼肌肌球蛋白进行提取和纯化,通过测定不同季节的两种淡水鱼肌球蛋白Ca2 -ATPase的热力学活化参数,从化学反应的热力学理论上证明了淡水鱼肌球蛋白的热稳定性与季节温度变化之间的关系.研究结果表明,草鱼和鲢鱼肌球蛋白Ca2 -ATPase的热力学活化参数活化能Ea、活化焓ΔH 和活化熵ΔS 伴随栖息环境温度的上升而增大,而活化自由能ΔG 的变化幅度很小,证明了肌球蛋白的热稳定性夏季鱼明显优于冬季鱼,而春季鱼和秋季鱼分别与夏季鱼和冬季鱼的相似.另一方面,ΔH 与ΔS 之间显示了良好的线性相关,证明了淡水鱼能够通过其肌肉蛋白质反应的活化焓与活化熵之间的互补效应,以应对自然界季节环境温度的变化.     

Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes

Using ⁵¹Cr-labelled P-815 mastocytoma cells as target cells and CS7BL/6 spleen cells sensitized against DBA/2 antigens as effector cells, it is shown that the variation in the observed specific ⁵¹Cr release over a broad range of experimental conditions can be explained on the basis of a simple physical model of the interaction process. The model assumes that a target cell can be destroyed only after contact with an effector cell, contact takes place on a random basis, one contact is sufficient, and that one effector cell can kill several targets with unchanged efficiency. The fraction of target cells destroyed (f) depends only on the incubation time (t), the number of effector cells (n) and a constant interaction probability (δ). Thus f = 1 ? e^?nδt. However, the experimental measurement, the fraction of ⁵¹Cr specifically released into the supernatant during the assay, may not be the same as the fraction of target cells destroyed because it takes considerable time for the releasable ⁵¹Cr to be released from a damaged target cell. This can be overcome experimentally by following the standard 37 °C incubation with a further incubation at 45 °C during which there are no new lytic events but all previously damaged target cells release the remainder of their releasable ⁵¹Cr. The model enables one to obtain accurate measurements of relative effector cell frequency over a broad range of experimental conditions.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

Response of catalase activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus

Catalase (CAT, EC 1.11.1.6) is an important enzyme in antioxidant defense system protecting animals from oxidative stress. Freshwater fish Oreochromis niloticus were exposed for 96 h to different concentrations of Ag(+), Cd(2+), Cr(6+), Cu(2+) and Zn(2+), known to cause oxidative stress, and subsequently CAT activities in liver, kidney, gill, intestine and brain were measured. In vivo, CAT was stimulated by all metals except Ag(+) in the liver and the highest increase in CAT activity (183%) resulted from 1.0 mg Cd(2+)/L exposure, whereas 0.5 mg Ag(+)/L exposure resulted in a sharp decrease (44%). In tilapia kidney, cadmium and zinc had no significant effects on CAT activity, whereas 0.1 mg Cr(6+)/L exposure caused a decrease (44%). Cadmium and zinc did not significantly affect the CAT activity in gill; however, 0.5 mg Ag(+)/L exposure caused an increase (66%) and 1.5 mg Cr(6+)/L exposure caused a decrease (97%) in CAT activity. All metals, except Cu(2+)(41% increase), caused significant decreases in CAT activity in the intestine. In brain, 1.0 mg Zn(2+)/L resulted in an increase in CAT activity (126%), while 1.5 mg Ag(+)/L exposure caused a 54% decrease. In vitro, all metals -- except Ag(+) and Cu(2+) in kidney -- significantly inhibited the CAT activity in all tissues. Results emphasized that CAT may be considered as a sensitive bioindicator of the antioxidant defense system.