微囊藻毒素与饮用水消毒副产物复合诱导草鱼淋巴细胞凋亡

针对饮用水中消毒副产物与微囊藻毒素的低剂量复合污染问题,采用离体细胞培养诱导方法,以草鱼淋巴细胞为暴露受体,研究了MCLR、MCRR两种微囊藻毒素和CHClBr₂、CHCl₂Br两种消毒副产物的单独与联合毒效应.结果表明:4种污染物在试验浓度下暴露2 h即能使草鱼淋巴细胞发生凋亡,且呈显著的剂量-效应关系;1 nmol·L^-1MCLR、MCRR分别和1～100 nmol·L^-1的CHCl₂Br、CHClBr₂的联合作用均表现为相加作用,并且均呈显著的剂量-效应关系.草鱼淋巴细胞凋亡率可作为一种有效的指标来评价微囊藻毒素及消毒副产物复合污染引起的细胞毒性.     

节球藻毒素对草鱼淋巴细胞凋亡毒性效应及机理

节球藻毒素是水华水体中发现的一种新型蓝藻毒素,鱼类很容易受到节球藻毒素的影响.本研究发现节球藻毒素能够体外诱导草鱼淋巴细胞发生凋亡.电镜观察从形态学上发现节球藻毒素诱导的草鱼淋巴细胞呈现明显的细胞质浓缩、染色质凝集以及边缘化等典型细胞凋亡特征.梯队状DNA进一步证实凋亡的发生.1、10和 100 μg·L^-1的节球藻毒素体外诱导草鱼淋巴细胞12 h后,凋亡率分别达到19.4%、31.6%和71.6%,具有明显的剂量效应特征.节球藻毒素诱导的草鱼淋巴细胞凋亡与胞内活性氧物质增加、线粒体膜电位下降、胞钙含量上升、Bcl₂基因下调和Bax基因上调有关;此外,Caspase-3和Caspase-9两种酶参与该凋亡过程.表明节球藻毒素能够通过线粒体通路诱导鱼体淋巴细胞发生凋亡.
     

微囊藻毒素对尼罗罗非鱼原代肝细胞致毒机理的探讨

采用离体细胞培养诱导方法,研究微囊藻毒素-LR(microcystin-LR,MC-LR)对尼罗罗非鱼(Oreochromis niloticus)原代肝细胞的毒性效应.尼罗罗非鱼原代肝细胞经10、50、150、500 μg/L MC-LR体外诱导24h后,单细胞微量凝胶电泳(SCGE)检测显示,与对照组相比处理组出现明显的彗星拖尾现象,说明MC-LR可引起尼罗罗非鱼肝细胞DNA的损伤,并随着剂量的增加,DNA的损伤程度增大.PI/Annexin V双染色流式细胞仪(FCM)检测表明MC-LR能明显引起肝细胞凋亡,与SCGE结果一致,且DNA损伤程度越大,细胞早期凋亡率越高,呈现明显的时间、剂量依赖性.本研究为进一步从分子、细胞水平阐明MC-LR的毒性以及致毒机理提供重要的理论依据.     

硒对亚硝酸钠导致的草鱼肝细胞氧化损伤的保护作用

以草鱼(Ctenopharyngodon idella)肝细胞(L8824)为研究对象,设置对照组、亚硝酸钠暴露实验组、亚硒酸钠孵育实验组和亚硒酸钠孵育后亚硝酸钠暴露实验组,探讨亚硒酸钠对不同浓度亚硝酸钠诱导L8824细胞氧化损伤及凋亡的保护作用。结果显示,亚硝酸钠暴露能抑制L8824细胞贴壁,导致细胞凋亡率增加。亚硝酸钠暴露引致L8824细胞的谷胱甘肽过氧化物酶(GPX)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性降低;gpx、sod和cat基因表达下调(P<0.05), DNA损伤诱导转录物3(ddit3)和bcl-2相关X蛋白(bax)基因表达上调(P<0.05)。亚硒酸钠(10μmol/L)孵育L8824对细胞形态和凋亡率无显著影响,但GPX、SOD和CAT活性上升,gpx、sod、cat、核因子E2相关因子2(Nrf2)和Kelch样环氧氯丙烷相关蛋白-1(keap1)基因表达上调(P<0.05)。亚硒酸钠孵育后亚硝酸钠暴露实验组,细胞凋亡率、GPX、SOD和CAT活性较对照组无显著变化,但B淋巴细胞瘤-2(bcl-2)基因表达显著上调(P<0...     

HIV-1 Tat蛋白通过增加线粒体ROS产生诱导人外周血B淋巴细胞凋亡

目的:探讨HIV-1 Tat蛋白对人外周血B淋巴细胞增殖、凋亡的影响及其机制。方法:采用流式细胞分选术分离HIV阳性患者外周血单核细胞的B淋巴细胞,分别转染pTat或pcDNA3.1各10μg(分别为pTat组与pcDNA3.1组),采用MTT实验检测细胞胞增殖情况,流式细胞术检测凋亡情况,DCHF-DA测定ROS水平,彗星试验检测细胞DNA损伤情况。结果:pTat组转染24h、48h的细胞增殖抑制率、细胞凋亡率及线粒体ROS水平均显著高于pcDNA3.1组(P0.05)。pcDNA3.1组细胞的DNA大部分呈圆形荧光团,无拖尾现象;pTat组的细胞DNA拖尾现象,呈现典型彗星图像。与pcDNA3.1组相比,pTat组细胞DNA尾长、尾部DNA比例均显著增加(P 0.05)。结论:HIV-1 Tat蛋白可能通过增加线粒体ROS产生,诱导DNA损伤,进而抑制人外周血B淋巴细胞增殖并促进其凋亡。     

miR-382-3p靶向RASA1基因调控骨关节炎软骨细胞增殖和凋亡

目的:探讨mi R-382-3p对骨关节炎软骨细胞增殖和凋亡的影响及其机制。方法:用100 ng/mL的脂多糖(LPS)处理软骨细胞,记为LPS组,以正常培养的软骨细胞作为正常对照(NC)组。mi R-NC、mi R-382-3p、anti-miR-NC、anti-miR-382-3p转染至软骨细胞中,记为mi R-NC组、mi R-382-3p组、anti-miR-NC组、anti-miR-382-3p组;将mi R-NC、mi R-382-3p、si-NC、si-RASA1转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-NC+LPS组、mi R-382-3p+LPS组、si-NC+LPS组、si-RASA1+LPS组;将mi R-382-3p分别与pcDNA-NC、pcDNA-RASA1共转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-382-3p+pcDNA-NC+LPS组、mi R-382-3p+pcDNA-RASA1+LPS组。实时荧光定量PCR(RT-qPCR)检测mi R-382-3p和Ras p21蛋白活化因子1(RASA1)m RNA表达水平;蛋白质印迹(Western blot)法检测RASA1、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡;荧光素酶报告实验检测mi R-382-3p和RASA1的靶向关系。结果:LPS诱导的软骨细胞中mi R-382-3p表达水平显著降低,RASA1表达水平显著升高,CyclinD1表达水平显著降低,Cleaved-caspase-3表达水平显著升高,细胞存活率显著降低,细胞凋亡率显著升高(P0.05)。过表达mi R-382-3p和敲减RASA1,LPS诱导的软骨细胞中CyclinD1表达水平显著升高,Cleaved-caspase-3表达水平显著降低,细胞存活率显著升高,细胞凋亡率显著降低(P0.05)。mi R-382-3p靶向调控RASA1,高表达RASA1部分逆转了mi R-382-3p高表达对LPS处理的软骨细胞增殖和凋亡的影响。结论:过表达mi R-382-3p促进软骨细胞增殖,抑制LPS诱导的软骨细胞凋亡,其机制可能与RASA1有关。     

X射线对人外周血淋巴细胞DNA损伤修复和凋亡的影响

目的 研究不同时间诱导X射线照射的淋巴细胞进入细胞周期DNA损伤修复与凋亡的影响.方法 X射线（0.5 Gy）作用于正常人外周血淋巴细胞,以照射后不同时间点（0、4 h）分别加入PHA并分成两组,即照射后0 h加PHA组（A组）和照射后4 h加PHA组（B组）,再分别培养0、0.5、2 h,用流式细胞术和免疫印迹法检测A组和B组γ-H2AX蛋白的表达,Annexin-V/PI法分析A、B两组的细胞凋亡率.结果 流式细胞术及免疫印迹结果均显示A组的γ-H2AX蛋白表达高于B组（P＜0.05）,且均先升高后降低.A组细胞凋亡率亦大于B组.结论 不同时间诱导被打击的淋巴细胞进入周期其可能发生DNA修复并同时伴随细胞凋亡的发生.     

左旋卡尼汀对脂多糖诱导小鼠肺微血管内皮细胞自噬和凋亡的影响*

目的:探讨左旋卡尼汀(LC)对脂多糖(LPS)损伤的小鼠肺微血管内皮细胞(PMVECs)的保护作用及自噬、凋亡的影响。方法:采用体外培养的小鼠PMVECs,分为对照组(Control组)、LPS组(10 μg/ ml,3、6、12、24 h)、LPS(10 μg/ ml,24 h)+LC(终浓度为2.5、5、10 μg/ml)(LC组)。Annexin V-FITC/PI双标记法检测细胞凋亡,细胞免疫荧光染色法检测自噬小体,Western blot法检测自噬相关蛋白LC3及凋亡蛋白Caspase-3的含量,CCK-8法检测细胞活力。结果:① 与Control组比较,LPS 6 h、12 h、24 h组PMVECs细胞活力显著受到抑制,细胞凋亡率、自噬蛋白LC3Ⅱ表达显著增高(P均＜0.01),LC3蛋白阳性表达。②与LPS 24 h组比较,各浓度LC组PMVECs细胞活力显著提高、自噬蛋白LC3II表达水平显著升高(P均＜0.01),而PMVECs凋亡率和凋亡蛋白Caspase-3表达水平均明显降低 (P＜0.05)。结论:LC具有提高LPS刺激的小鼠PMVECs活性、促进PMVECs自噬、抑制凋亡的作用。     

高原低氧免疫损伤及其干预措施的研究

目的:探讨高原低氧损伤免疫系统的特征及其可能机制,研究高原低氧免疫损伤的干预措施。方法:测定低氧暴露不同时间小鼠免疫器官指数、外周血和免疫器官T淋巴细胞亚群的变化;观察小鼠免疫器官淋巴细胞凋亡率及小鼠肺脏和肾脏病理学改变。采用预防给药方式,研究中药组方对低氧免疫损伤小鼠的干预作用。结果:①模拟海拔8000m低氧暴露8h后,小鼠胸腺CD4+CD8+细胞数显著下降,CD4+CD8-、CD4-CD8+细胞数显著增加(P0.01);低氧暴露3d后,外周血CD4+细胞明显减少(P0.05),CD4+/CD8+比值显著降低(P0.05),胸腺CD4+CD8+细胞数进一步下降,CD4+CD8-、CD4-CD8+细胞数进一步增加,小鼠脾脏、胸腺淋巴细胞晚期凋亡和坏死率均显著增加(P0.05);低氧暴露6d后,小鼠脾指数显著性增加(P0.01);胸腺指数显著性降低(P0.01),脾CD4+、CD8+细胞数显著降低(P0.01),脾脏和胸腺淋巴细胞晚期凋亡率和坏死率进一步增加(P0.01),活细胞率显著降低(P0.01),脾脏淋巴细胞早期凋亡率显著增加(P0.01)。整个低氧暴露过程中外周血CD8+无显著性变化。②新复方党参、香杞多糖、二者联合应用均能显著增加低氧免疫损伤小鼠外周血CD3+、CD4+、脾脏CD4+的细胞水平(P0.01,P0.05),对脾脏CD8+细胞水平没有显著影响。香杞多糖及其与新复方党参联合应用均能进一步降低胸腺CD4+CD8+,进一步增加CD4+CD8-的细胞水平(P0.01),未见对CD4-CD8+细胞水平的影响;新复方党参对低氧免疫损伤小鼠胸腺没有显著性影响。结论:模拟海拔8000m低氧暴露后小鼠外周发挥免疫作用的淋巴细胞数减少可能与低氧暴露早期淋巴细胞凋亡率和坏死率增加和肺脏淋巴细胞分布增多有关。新复方党参和香杞多糖作为低氧免疫损伤干预措施,具有一定发展前景。     

水体铅暴露下草鱼幼鱼游泳行为和代谢响应

为探讨重金属铅(Pb)污染对鱼游泳能力和代谢的影响,在实验室条件下,分析了20℃时4组Pb~(2+)浓度(0、0.75、2.24和3.73 mg·L~(-1))暴露对草鱼(Ctenopharyngodon idella)幼鱼临界游泳速度(Ucrit)和运动耗氧率(MO_2)的影响。结果表明:与对照组比较,Pb~(2+)浓度为3.73 mg·L~(-1)时草鱼幼鱼Ucrit显著下降(P0.05),Ucrit为对照组的91.22%;草鱼幼鱼游泳速度与耗氧率均线性相关;随着Pb~(2+)浓度增加,草鱼幼鱼最大和最小耗氧率差值先增加后降低,Pb~(2+)浓度为3.73 mg·L~(-1)时这一差值下降了17.68%;低浓度Pb~(2+)暴露引起运动过程中耗氧利用效率降低,可能是导致草鱼幼鱼游泳能力下降的主要原因;通过草鱼耗氧和行为对急性Pb~(2+)暴露的响应研究,为河流Pb~(2+)生态风险评价及控制提供参考。     

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

环境毒物对金黄色葡萄球菌毒性影响的研究

采用毒物在营养琼脂中垂直扩散方法,通过测定毒物在营养球脂中抑制金黄色葡萄球菌生长产生蓝色抑菌带的长度,研究Hg、Cr^6 、Pb、CN^—、As、NO2^—、F^—及苯酚对金黄色葡萄球菌的毒性影响。结果表明：受试毒物的浓度与抑菌带有相关性,相关系数具显著意义;对毒物的敏感性为Cr^6 ＞Hg＞As＞CN^—＞Pb＞NO2^—＞苯酚＞F—;多种毒物共同作用其毒性影响增加。     

Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.     

鲤的微卫星引物对草鱼基因组分析适用性的初步研究

运用微卫星DNA-聚合酶链反应(STR—PCR)基因分型技术,选取已发表的28对鲤的微卫星引物,探讨鲤的引物用于草鱼基因组微卫星分析的可能性。通过优化PCR反应条件,消除了影子带和异源核酸双链分子两类微卫星相关假阳性带对STR—PCR分析的干扰。在此基础上,筛选出7对引物可在湘江野生草鱼基因组中扩增出特异性条带,占总数的25％;其中的4对引物(约占总数的14．3％)在8尾湘江野生草鱼小群体中即检测到了个体间等位基因的多态性。这些初步的结果表明鲤的微卫星引物可以用于草鱼基因组的分析。     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.