共查询到20条相似文献，搜索用时 374 毫秒
1.
一种用磷酸钙法高效转染HEK 293T细胞方法的建立 总被引：1，自引：0，他引：1
利用磷酸钙转染法将lacZ-pShuttle质粒导入HEK 293T细胞中,实验证明,pH值、DNA纯度、转染后培养时间和甘油休克时间是影响转染效率的重要因素.转染后培养6h进行甘油休克效果最佳,比培养0h直接进行休克转染效率升高584.4%;甘油休克时间为4.5min时转染效果最佳,比不用甘油休克转染效率升高130.8%.通过对影响转染的几个条件进行优化,建立了1种可以高效转染HEK 293T细胞的方法,且简便易行. 相似文献
2.
肌肉生长抑制素抑制动物肌肉的生长发育.根据本实验室克隆的大黄鱼（Pseudosciaena cro-cea）肌肉生长抑制素（MSTN）基因编码序列设计引物,用RT-PCR扩增目的片段,构建MSTN-pET-28 a重组表达质粒,将其转化到大肠杆菌BL21上并用1.0 mmol/dm3IPTG诱导表达,SDS-PAGE电泳检测该目的蛋白大小约为44 kDa,与理论值大小符合.用0.25 mol/dm3KCl染色后从凝胶中切取该蛋白且回收.该回收蛋白与弗氏佐剂等量混合后注射ICR小鼠制备多克隆抗体,并用Westernblot检测该抗体.免疫印迹结果在44 kDa的位置上出现棕色条带,表明大黄鱼肌肉生长抑制素的多克隆抗体制备成功.这为今后对肌肉生长抑制素功能的研究奠定了基础. 相似文献
3.
目的：观察银杏提取物EGb761对大鼠视神经损伤后视网膜神经节细胞自噬活性及自噬流的影响，并探讨其作用机制。方法：将48只SD大鼠随机分为EGb761组和对照组，分别给予EGb761（150mg/kg·d）和0.9%氯化钠注射液灌胃，均每天1次，直到处死。分别于损伤后第7天、第14天取材，采用免疫组化检测2组视网膜神经节细胞自噬相关蛋白LC3B、自噬标记蛋白p62及自噬基因Beclin-1及mTOR通路蛋白p-mTOR、p-S6表达水平，腺病毒GFP-mRFP-LC3荧光瞬时转染技术检测自噬流表达。结果：与对照组相比，EGb761组LC3B表达水平升高，p62、p-mTOR和p-S6蛋白表达水平均下降（均P<0.05）；2组Beclin-1差异无统计学意义（P＞0.05）；2组GFP和mRFP荧光点数比较，EGb761组的红点多于对照组，黄点少于对照组（均P<0.05）。结论：EGb761能够促进大鼠视神经损伤后视网膜神经节细胞自噬流，其机制可能与促进mTOR信号通路中的相关蛋白表达有关。 相似文献
4.
micro RNA为短链非编码RNA,通过与靶基因3′UTR序列互补在转录后水平发挥作用。已有研究表明,micro RNA在红细胞发生过程中起着重要的调控作用。南极冰鱼是目前已知的唯一仅具有无功能性血红细胞的脊椎动物。前期研究提示,独角雪冰鱼(Chionodraco hamatus)头肾中高表达的micro RNAs可能抑制着冰鱼红血球的发生。本研究针对南极冰鱼头肾中高表达的miR-7132,运用斑马鱼显微注射、双荧光素酶报告系统,并结合靶基因预测等手段研究了miR-7132对血红细胞发生的作用机制。结果表明:斑马鱼胚胎注射miR-7132后,固蓝染色显示斑马鱼胚胎红细胞中血红蛋白的表达显著下降,这表明miR-7132的过表达抑制了血红细胞生成。通过转录组数据结合比对冰鱼的全基因组序列,获得了独角雪冰鱼血红细胞生成的血红素生物合成的限速酶(5′-aminolevulinate synthase 2,ALAS2)基因3′UTR序列。构建含ALAS2基因3′UTR序列的双荧光素酶报告质粒,并与miR-7132共转染293T细胞检测荧光素酶活性变化;构建包含绿色荧光蛋白(green fluorescent protein,GFP)的表达质粒与miR-7132共同注射斑马鱼胚胎,检测GFP荧光强度与蛋白表达量变化。结果显示,转染miR-7132的293T细胞荧光素酶相对活性显著降低,ALAS2基因是miR-7132的一个靶基因。斑马鱼体内注射miR-7132的GFP荧光强度显著降低,且GFP蛋白表达量显著减少。本研究揭示了miR-7132对血红细胞生成的抑制作用,miR-7132通过抑制ALAS2基因的表达而抑制南极冰鱼血红细胞的发生。 相似文献
5.
中国对虾细胞色素P450基因CYP4原核表达条件优化 总被引：1，自引：0，他引：1
根据克隆得到的中国对虾（Fenneropenaeus chinensis）CYP4基因开放阅读框设计引物,构建了中国对虾CYP4基因原核表达载体p28a-CYP4,并对重组菌株Rosetta／28a-CYP4的原核表达条件进行了优化。结果表明：p28a—CYP4转化Rosetta后可实现CYP4基因的原核表达,SDS—PAGE分析显示其在56．0kDa处有显著诱导条带;通过对诱导温度、IPTG浓度、诱导时机（OD600）及诱导时间的优化表明,重组菌株Rosetta／p28a-CYP4的最佳诱导温度为37℃,最佳IPTG浓度为1．2mmol／L,最佳诱导时机及诱导时间分别为0．59和6h。 相似文献
6.
目的：通过脂多糖（LPS）干预单核细胞（THP-1）源性泡沫细胞制备高胆固醇模型，探讨右归丸含药血清对THP-1源性泡沫细胞胆固醇流出与炎症因子表达的影响。方法：利用LPS培养THP-1源性泡沫细胞诱导24h制备高胆固醇细胞模型，给予右归丸含药血清小、中、大剂量干预。通过比色法检测干预后的THP-1源性泡沫细胞内总胆固醇（TC）、游离胆固醇（FC）和胆固醇酯（CE）的含量；酶联免疫吸附法测定细胞内的细胞介素1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）的含量；逆转录-聚合酶链反应法检测细胞NF-κB p65、SREBP1c、ABCA1mRNA的含量和蛋白表达。结果：10 ng/ml LPS培养THP-1源性泡沫细胞成功复制了高胆固醇细胞模型。右归丸大、中、小剂量组THP-1源性泡沫细胞内TC、FC、CE、IL-1β、IL-6、TNF-α、NF-κB p65、SREBP1c、ABCA1mRNA含量以及NF-κBp65、SREBP1c、ABCA1mRNA蛋白表达水平与正常组比较，差异均有统计学意义（P＜0.05或P<0.01）。结论：右归丸可能是通过抑制NF-κB p65和SREBP1c基因表达而调节血脂，同时通过抑制炎症因子的释放来预防动脉粥样硬化，为临床预防和治疗动脉粥样硬化提供了新的靶点。 相似文献
7.
以大黄鱼（Larimichthys crocea）为研究对象,研究急性、慢性低盐度胁迫对大黄鱼存活状况及非特异性免疫酶活力的影响。结果表明：急性低盐（15、8）胁迫下,在7 d的实验周期中,大黄鱼肝脏的超氧化物歧化酶（superoxide dismutase, SOD）活力呈先上升后下降的趋势,在第1天显著上升（p<0.05）后逐渐下降至显著低于对照组水平;肝脏的过氧化氢酶（catalase, CAT）活力整体呈先下降后上升再下降的趋势,在第1天显著下降后开始升高,第3天后开始下降,至第7天时CAT活力仍显著低于对照组水平;血清中的酸性磷酸酶（acid phosphatase, ACP）活力呈下降后逐渐升高的趋势,在第1 天时显著下降后逐步升高,至第7天时仍显著低于对照组;而血清中的碱性磷酸酶（alkaline phosphatase, ALP）活力在实验开始的第1天到第3天逐渐升高,且均显著高于对照组,至第7天时开始下降至对照组水平;血清中的溶菌酶（lysozyme, LZM）含量呈波动变化,整体呈先上升后下降趋势。慢性低盐（8）养殖14 d后,大黄鱼的各项非特异性免疫酶活力均与对照组无显著性差异（p>0.05）。此外,实验周期内所有组别大黄鱼均未出现死亡,仅急性低盐胁迫组大黄鱼的活动和摄食受到有限影响。大黄鱼对慢性降盐度养殖有较高的耐受能力,而盐度骤降会显著影响大黄鱼的非特异性免疫,实际生产中应避免养殖环境盐度的剧烈变化。 相似文献
8.
大黄鱼(Pseudosciaena crocea)热激蛋白的基因克隆、原核表达及抗血清的制备 总被引：1，自引：1，他引：1
应用trizol裂解法提取象山港网箱养殖大黄鱼肝脏总RNA,采用RTPCR获得大黄鱼cDNA;通过设计特异引物进行PCR扩增,将PCR产物转化到质粒并直接测序,得到1.7kb的目的基因;同时将此目的基因连接到表达质粒pSBET中,在大肠杆菌BL21中进行诱导表达以及表达产物的分析。克隆基因的表达产物经SDSPAGE分析表明,HSP基因的蛋白分子量为60kDa左右,与阅读框的编码大小一致;表达蛋白经纯化后免疫小鼠制备抗血清。Westernblotting检测结果表明,制备的抗血清与HSP蛋白起较强的特异性反应。HSP基因的系统进化分析表明,大黄鱼与非洲爪蟾最为接近,从侧面印证了脊椎动物中两栖纲动物与鱼纲动物的亲源关系。通过大黄鱼HSP基因的提取和分析,可为以后制备出核酸探针、筛选和克隆出一批具有优良性状的基因、构建基因文库等研究工作奠定基础。 相似文献
9.
对虾传染性皮下及造血组织坏死病毒衣壳蛋白基因的克隆表达及抗体制备 总被引：2，自引：0，他引：2
本文根据GenBank登录的对虾传染性皮下及造血组织坏死病毒(IHHNV)的全基因组序列(NC-002190),设计出主要结构蛋白基因的相应引物,从厦门发病的对虾中,提取DNA,以此为模板,利用PCR扩增目的基因.再将目的基因分别与pGEX-6p1载体和pET-28a载体连接,构建重组表达载体pGEX-Ⅳ、pET-Ⅳ,重组子经酶切和测序鉴定后导入表达型大肠杆菌BL21,IPTG诱导表达,SDS-PAGE检测,大小约为62KDa和41KDa,与预测大小一致.将pET-Ⅳ表达重组蛋白经纯化,免疫小鼠,Western免疫印迹反应结果表明其抗体均可与pGEX-Ⅳ和pET-Ⅳ表达的蛋白发生特异性反应.本研究的结果为对虾IHHNV的快速检测及其免疫提供了研究基础. 相似文献
10.
在已构建重组质粒pGEM—vp28的基础上,通过NcoⅠ,HindⅢ双酶切质粒pGEM—vp28和表达载体质粒pET-30a,分别获得携带限制性内切酶粘性末端的vp28基因片段和表达载体质粒片段,两片段经T4DNA连接酶连接获得了重组表达质粒pET30a—vp28,将此重组质粒导入表达宿主菌Escherichia coli BL21中,用IFIG诱导表达4h后,其表达产物在SDS—PAGE和Western blot中的显示的特异性条带大小约27ku,与预期的大小相近。 相似文献
11.
Oxidative stress induced by xenobiotic compounds has been studied using primary hepatocytes of juvenile European flounder (Platichthys flesus L.) caught in a low polluted area of the German Bight, Tiefe Rinne (Landwüst et al., 1996.). Cells were exposed to known oxidative stressors such as hydrogen peroxide and benzo[a]pyrene (B[a]p) in various concentrations (50 and 100 microM) up to 6 days. Cell mortality was determined using fluorescent ethidium homodimer-1 and calcein AM. Oxidative stress response was measured by image analysis using dihydrorhodamine 123, which is converted to fluorescent rhodamine 123 in the presence of intracellular ROS. Oxyradical formation was initiated already after 2 h of exposure to low concentrations of B[a]p and hydrogen peroxide. Probably due to a membrane stabilising effect of the serum factors the addition of fetal bovine serum to the culture medium had a protecting influence on the hepatocytes and resulted in (1) an increased cell viability and (2) reduced formation of intracellular ROS during exposure. In conclusion, the assay is a sensitive tool for testing the potential of various xenobiotics to induce oxidative stress in living hepatocytes. 相似文献
12.
The aim was to evaluate the antioxidant defences and the occurrence of oxidative damage in Posidonia oceanica under a stress situation due to the epiphytism of the invasive macroalgae Lophocladia lallemandii. P. oceanica leaves were collected in the absence (control station) and in the presence of the epiphytic algae L. lallemandii and the antioxidant enzyme activities, markers of oxidative damage, and hydrogen peroxide production were determined. Antioxidant enzyme - catalase, glutathione peroxidase and superoxide dismutase - activities were significantly higher in Posidonia epiphytized by L. lallemandii. Malondialdehyde, protein carbonyl derivates, and glutathione levels were also higher in L. lallemandii epiphytized P. oceanica leaves compared to control samples. The production of hydrogen peroxide was also significantly increased when Posidonia was epiphytized by L. lallemandii. The invasion of P. oceanica meadows by L. lallemandii appeared to induce oxidative stress in the seagrass as evidenced by increased levels of oxidative stress markers and antioxidant defences. 相似文献
13.
Kimette Radtke Robert W. Byrnes Pamela Kerrigan William E. Antholine David H. Petering 《Marine environmental research》1992,34(1-4)
It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H2O2 in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H2O2 outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H2O2 that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H2O2, measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide. 相似文献
14.
苯并芘对褐菖鲉肝细胞DNA交联的影响 总被引：2，自引：0，他引：2
以褐菖鲉（Sebastiscus marmoratus）为实验材料,设定苯并芘[B（a）P]的注射剂量梯度分别为0.00、0.05、0.50、5.00 mg/kg,分别于实验的第0、3、6天对每一条鱼进行腹腔注射,于第1、4、7天取样,测定胆汁中的B（a）P及其代谢物含量.采用荧光检测法检测B（a）P染毒后褐菖鲉肝细胞DNA-DNA交联（DDC）交联系数的变化,并用KC l-SDS沉淀法检测DNA蛋-白质交联（DPC）交联系数的变化.结果表明：（1）经注射染毒后鱼体胆汁中的B（a）P及其代谢物含量蓄积程度存在着明显的剂量效应和时间效应;（2）B（a）P的注射剂量分别为0.05、0.50 mg/kg时各时间点褐菖鲉肝细胞DDC交联系数均无显著增大（p〉0.05）,在5.00 mg/kg剂量组,随着时间延长DDC交联系数逐渐增大,第4天即有显著差异（p〈0.05）,第7天差异极显著（p〈0.01）;（3）B（a）P诱导DPC的趋势与DDC类似,但0.5 mg/kg剂量组在第7天DPC交联系数即出现显著增大（p〈0.05）.这表明B（a）P对DPC的诱导高于DDC.这为进一步研究B（a）P的遗传毒性提供了依据,并为探讨B（a）P致癌的作用机理提供了新的信息. 相似文献
15.
Leão JC Geracitano LA Monserrat JM Amado LL Yunes JS 《Marine environmental research》2008,66(1):92-94
Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystisaeruginosa strain RST9501 ( approximately 2 microg/mL microcystins, MC); 10 were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and 10 were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC. 相似文献
16.
为阐释不同浓度苯并（a）芘[B（a）P]对杂色鲍（Haliotis diversicolor）的毒性效应,以0.02、0.04和0.08 mg/dm^33个质量浓度的B（a）P对杂色鲍进行水体暴露胁迫,7d后检测肌肉、外套膜、鳃、性腺、肾和肝胰腺的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽S-转移酶（GST）和谷胱甘肽过氧化物酶（GPx）活性及还原型谷胱甘肽（GSH）含量.结果显示：0.02 mg/dm^3的B（a）P暴露对杂色鲍抗氧化系统的影响不明显,而0.04、0.08 mg/dm^3的B（a）P则显著抑制了SOD、CAT和GPx酶活性以及GSH含量,同时对GST酶活性具有显著的诱导作用;实验还发现,不同组织抗氧化酶活性对B（a）P胁迫的敏感性响应存在较大差异,其中鳃、肾脏和肝胰腺的敏感性响应明显高于肌肉和外套膜.上述结果表明,苯并（a）芘暴露对杂色鲍抗氧化酶活性的影响具有明显的剂量-效应关系及组织差异性,杂色鲍的鳃、肾脏或者肝胰腺的抗氧化酶响应多环芳香烃胁迫的更敏感,可作为B（a）P污染的生物标志物. 相似文献
17.
利用流式细胞仪（FCM）技术检测了杂色鲍血细胞在体内和体外对不同含量荧光微球悬液的吞噬率.其结果表明体内注射法测定吞噬率的方法不可行.在体外吞噬中,当荧光微球含量为血细胞含量的68.2倍时吞噬率达98.43%±1.27%,随着荧光微球含量的降低血细胞吞噬率也随着降低.当荧光微球含量为血细胞含量的6.82倍时吞噬率为63.45%±6.05%,以这一含量进行血细胞吞噬率的测定比较恰当.利用这一含量测定了正常鲍与经过低盐（S=18）海水胁迫24 h后鲍的血细胞吞噬率的变化,正常鲍的吞噬率为68.71%±10.05%,经低盐海水胁迫后鲍的吞噬率为49.49%±10.18%,吞噬率出现了显著的下降（p〈0.05）.实验结果表明利用流式细胞仪技术相比传统的显微镜镜检记数法检测杂色鲍血细胞吞噬率更为方便可靠. 相似文献
18.
高效降解有机物和促藻生长菌株的分离和筛选 总被引：3，自引：0，他引：3
本实验采用对虾饲料浸出液培养基从对虾健康养殖池中富集并分离出60株优势菌．通过对蛋白质、淀粉和CODMn的降解能力及对小球藻（Chlorella sp．）生长的影响等指标的检测，筛选出5^#、9^#和24^#共3株能高效降解有机物和促藻生长的菌株．10d内，其对CODMn的去除率均在40％以上．在有机培养液中，这3株菌对小球藻生长均有显著促进作用（p〈0．05），经过7d培养，藻细胞密度分别比对照菌提高了92．3％、98．1％、32．7％．在无机培养液中，9^#菌对小球藻生长有明显的促进作用（P〈0．05），经过7d培养，藻细胞密度比对照菌提高了77．3％．经初步鉴定5^#、9^#和24^#这3株菌分别属于弧菌属（Vibrio）、假单胞菌属（Pseudomonas）和芽孢杆菌属（Bacillus）的细菌． 相似文献
19.
The concentration of hydrogen peroxide was measured in waters off the coast of Peru during June and July 1983. The study period coincided with the end of the 1982/83 El Niño and the onset of coastal upwelling. Depth profiles of hydrogen peroxide concentration exhibit surface maxima and decrease with depth to the base of the mixed layer. Surface peroxide concentrations ranged from 0.8 to 5 × 10?8 M. Below the mixed layer hydrogen peroxide was below the detection limit (5 × 10?9 M). Diel variations were observed, with surface peroxide levels increasing during the day and decreasing at night. The nearshore station exhibited lower hydrogen peroxide concentrations than offshore stations, a reversal of the trend found in other coastal regions. This is attributed to the lack of coastal vegetation and runoff, and to active coastal upwelling of deeper water with low hydrogen peroxide concentrations. 相似文献
20.
Levels of cellular glutathione and metallothionein affect the toxicity of oxidative stressors in an established carp cell line. 总被引：4，自引：0，他引：4
J Wright S George E Martinez-Lara E Carpene M Kindt 《Marine environmental research》2000,50(1-5):503-508
The comparative toxicity of a variety of oxidative stressors was studied in the epithelioma papulosum cyprini line from carp using the neutral red cytotoxicity assay. LC50's decreased in the order t-butylhydroperoxide > hydrogen peroxide > diquat > paraquat. The cytotoxicity of hydrogen peroxide was significantly reduced when the cells were grown in L-15 medium rather than MEM and this could be attributed to elevated cellular glutathione and metallothionein levels and higher activities of GSH-dependent detoxification systems. The protective effect of metallothionein in radical scavenging was demonstrated by decreased toxicity of the redox-cycling toxicants, diquat and menadione after metallothionein levels had been pre-induced by Cd-exposure. This study demonstrates the relationship between toxic effects of oxidative stressors and expression of detoxification systems in fish. 相似文献