慧星试验检测洛克沙胂对CHL细胞的DNA损伤

利用碱性单细胞凝胶电泳技术研究洛克沙胂对中国仓鼠肺细胞(CHL)的DNA损伤影响。洛克沙胂分为10、100、500、1000mg/L剂量组,分别以磷酸盐缓冲液和10mg/L亚砷酸钠为对照组,经3、6、12、24、48h暴露后进行单细胞凝胶电泳。结果表明,慧星试验参数尾DNA含量、慧星全长、慧尾长、尾距和Olive尾距等表现出剂量-效应、时间-效应关系,不同剂量洛克沙胂、不同暴露时间下对CHL细胞有不同程度的DNA损伤。     

镉致大鼠BRL 3A细胞DNA及线粒体损伤

为了研究镉对大鼠肝细胞系(BRL 3A细胞)DNA及线粒体损伤的作用,本研究选用0、10、20、40 μmol/L的醋酸镉分别作用于BRL 3A细胞12h,利用四氮唑蓝比色分析法(MTT法)测定细胞存活率,显微镜观察各组细胞形态,通过彗星试验法检测细胞DNA的损伤,用透射电镜观察细胞线粒体超微结构,并检测细胞Caspase 3,9的活力.结果显示,随镉浓度的增大(10～40 μmol/L),BRL 3A细胞存活率降低,拖尾率、尾长、尾部DNA含量、细胞Caspase 3,9的活力和线粒体变形肿胀以及空泡现象均呈增加趋势;表明镉可致BRL 3A细胞DNA及线粒体损伤,并呈剂量依赖关系.     

改良彗星实验检测黄曲霉毒素B_1致雏鸭DNA损伤

试验旨在通过改良彗星实验检测黄曲霉毒素B1(aflatoxi-B1,AFB1)对雏鸭肝细胞DNA损伤的影响.锥鸭经AFB1灌胃染毒,2 h后分离肝细胞,并通过改良彗星实验测定DNA损伤.结果显示,AFB1能够导致雏鸭肝细胞DNA损伤,表现为尾长、尾部DNA百分含量、尾矩、Olive尾矩等彗星参数与空白和溶剂对照组相比显著增加(P<0.05).表明改良彗星实验能够用于AFB1导致肝细胞DNA损伤的检测,试验还提示,在体肝细胞彗星实验能够作为雏鸭AFB1暴露的遗传毒性标志物.     

The Comet assay for detection of DNA damage in canine sperm

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno‐, terato‐ or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A—median: 31.55%, interquartile range (IQR): 30.18–38.01; group B—median: 0.90%, IQR: 0.65–1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = ?.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non‐normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.     

芪苓制剂多糖对环磷酰胺所致免疫损伤小鼠血清SOD和MDA的影响

探讨芪苓制剂多糖对环磷酰胺所致免疫损伤小鼠血清SOD和MDA的影响。将75只体重18-22 g的雄性昆明小鼠随机均分为5组,即空白对照组、环磷酰胺(CY)组和芪苓制剂多糖低、中、高剂量组,芪苓制剂多糖3个剂量组小鼠分别灌服低(200 mg/kg)、中(400 mg/kg)、高(600 mg/kg)剂量芪苓制剂多糖,空白对照组和CY组分别灌服等量的蒸馏水,1次/d,连续19 d。试验第20天,空白对照组小鼠腹腔注射生理盐水,其余4组均腹腔注射100 mg/kg CY,24 h后,各组小鼠摘除眼球采血,检测小鼠血清SOD活性和MDA含量。结果表明,与CY组相比,芪苓制剂多糖3个剂量组小鼠血清SOD活性均极显著升高(P<0.01),MDA含量均极显著降低(P<0.01)。结果表明,芪苓制剂多糖能改善环磷酰胺所致免疫损伤小鼠的抗氧化能力。     

F-2毒素致体外培养大鼠睾丸支持细胞DNA损伤

为探讨F-2毒素对雄性生殖机能的影响,取大鼠睾丸支持细胞进行体外培养,运用单细胞凝胶电泳技术检测51、0、20和40 mg/L的F-2毒素攻毒后24 h支持细胞DNA的损伤情况。结果显示,F-2毒素在一定浓度范围内对体外培养的支持细胞DNA产生损伤作用,且受损程度随着F-2毒素攻毒剂量而升高,具有明显量效关系。除5 mg/L剂量组外,其余各组的细胞受损率、彗星尾长和DNA损伤程度与阴性对照组相比差异极显著（P〈0.01）,表明F-2毒素对体外培养的大鼠睾丸支持细胞有毒性作用,能够损伤细胞DNA。     

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H₂O₂ and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H₂O₂ (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.     

Managing crop damage caused by house mice (Mus domesticus) in Australia

A large-scale outbreak of the house mouse populations occurs in grain growing in Australia on average once every four years. High densities of mice cause major yield losses to cereal crops, and low to moderate densities of mice also cause some losses. Several predictive models based on rainfall patterns have been developed to forecast mouse density. These models carry some uncertainty and the economic value of basing management actions on these models is not clear. Baiting is the most commonly used method and zinc phosphide and other rodenticide bait are effective in reducing up to 90% of mouse populations. Ecologically-based best farming practice for controlling mice has recently been developed on the basis of long-term field studies of mouse populations. No effective biological control method has been developed for mice. However, grain growers still cannot make economically rational decisions to implement control because they do not know the pest threshold density (D_T) above which the economic benefits of control exceed the economic costs of control. Applied predator-prey theory suggests that understanding the relationship between mouse density and damage is the basis for determining D_T. Understanding this relationship is the first research priority for managing mouse damage. The other research priority is to develop a reliable method to estimate unbiased mouse density.     

Betulinic acid prevents alcohol-induced liver damage by improving the antioxidant system in mice

Betulinic acid (BA), a pentacyclic lupane-type triterpene, has a wide range of bioactivities. The main objective of this work was to evaluate the hepatoprotective activity of BA and the potential mechanism underlying the ability of this compound to prevent liver damage induced by alcohol in vivo. Mice were given oral doses of BA (0.25, 0.5, and 1.0 mg/kg) daily for 14 days, and induced liver injury by feeding 50% alcohol orally at the dosage of 10 ml/kg after 1 h last administration of BA. BA pretreatment significantly reduced the serum levels of alanine transaminase, aspartate transaminase, total cholesterol, and triacylglycerides in a dose-dependent manner in the mice administered alcohol. Hepatic levels of glutathione, superoxide dismutase, glutathione peroxidase, and catalase were remarkably increased, while malondialdehyde contents and microvesicular steatosis in the liver were decreased by BA in a dose-dependent manner after alcohol-induced liver injury. These findings suggest that the mechanism underlying the hepatoprotective effects of BA might be due to increased antioxidant capacity, mainly through improvement of the tissue redox system, maintenance of the antioxidant system, and decreased lipid peroxidation in the liver.     

女贞子多糖抑制MDA缓解CCl_4致小鼠肝损伤

本研究应用女贞子多糖给小鼠饮水,探讨其对四氯化碳诱导肝损伤的保护作用。试验分为对照组、模型组、女贞子多糖高、中、低剂量组和阳性药组,通过检测各组小鼠的生长性能、肝脏指数、血清生化指标和丙二醛(MDA)的表达量,综合评估女贞子多糖的保肝作用。结果表明:与模型组相比较,饮水中添加不同剂量的女贞子多糖对小鼠生长性能无影响(P>0.05),但可以显著缓解四氯化碳所致的肝脏指数(P<0.05)、肝功能指标(P<0.05)和肝脏血清MDA含量(P<0.05)。饮水中添加女贞子多糖可以有效缓解小鼠化学性肝损伤,这可能与调节肝脏MDA的表达,抑制脂质过氧化反应有关。     

禽多杀性巴氏杆菌ompa DNA疫苗在小鼠体内免疫效果试验

应用PCR技术扩增获得禽多杀性巴氏杆菌的ompa基因片段,克隆到pUCm-T载体,再亚克隆到真核表达质粒载体pCDNA3.1(+)上,构建重组质粒pcA,体外转染Vero细胞,RT-PCR和间接免疫荧光试验检测其转录表达情况。动物免疫分为3组:pCDNA3.1(+)组、PBS对照组和pcA组,每组16只BALB/c小鼠,pCDNA3.1(+)组和pcA组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2周。间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,三免2周后检测脾淋巴细胞IFN-γ分泌情况。强毒攻击,计算小鼠存活数目及保护率。结果显示,间接免疫荧光试验和RT-PCR检测结果均表明pcA可在体外培养的Vero细胞中表达目的蛋白。动物免疫后,pcA组免疫小鼠血清抗体水平持续上升,与pCDNA3.1(+)组和PBS组相比差异极为显著(P<0.01)。经提取的禽多杀性巴氏杆菌总外膜蛋白(Omps)刺激后,pcA组的刺激值(SI值)与pCDNA3.1(+)组及PBS免疫组相比均差异显著(P<0.05)。脾细胞产生的IFN-...     

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.     

不同剂量小鹅瘟病毒VP3基因疫苗肌肉注射诱导小鼠细胞免疫

将小鹅瘟(Gosling plagne,GP)VP3基因疫苗(pcDNA-GPV-VP3)分别按每只50、100、200 μg肌肉注射免疫BALB/c小鼠,以pcDNA3.1( )和生理盐水为对照,于免疫后7、14、21、28、35、63、105 d采血用淋巴细胞转化实验(MTT法)和流式细胞仪(FACS)分别检测小鼠外周血T淋巴细胞转化效果和CD4 、CD8 T淋巴细胞动态变化.结果表明,pcDNA-GPV-VP3免疫小鼠能够诱导机体产生良好的细胞免疫应答.50、100 μg pcDNA-GPV-VP3免疫后小鼠外周血T淋巴细胞对ConA刺激的反应显著或极显著高于200 μg组、空载体和生理盐水对照组,且以100 μg组最优,而200 μg组的转化效果比空载体组差;100 μg pcDNA-GPV-VP3免疫小鼠后所诱导的CD4 T淋巴细胞免疫功能最强,50 μg组次之;50 μg pcDNA-GPV-VP3免疫小鼠后所诱导的CD8 T淋巴细胞免疫功能最强,100 μg组次之.     

The association of Hsp90 expression induced by aspirin with anti-stress damage in chicken myocardial cells

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.     

Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation

Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288–299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.     

枸杞多糖对己烯雌酚生殖损伤仓鼠抗氧化能力及生殖激素水平的影响

为探讨枸杞多糖（LBP）对己烯雌酚（DES）致成年雄性仓鼠生殖损伤的保护作用,66只成年雄性仓鼠随机分为空白对照组（A）、DES组（B）、LBP组（C、D、E）及VC＋VE组（F）。除对照组注射橄榄油外,其余5组皮下注射DES,连续用药7d,同时C、D、E组分别灌服1,10,50mg/kg的LBP,F组灌服100mg/kg的VC及200IU/kg的VE,A、B组灌服等剂量的生理盐水,于末次给药24h后取材。摘取睾丸、附睾和精囊腺测质量并观察睾丸组织结构,检测血清中SOD、GSH-Px、MDA及T、LH、FSH、E2的水平。结果显示,DES能显著降低仓鼠体质量及生殖器官质量,而10,50mg/kg LBP及维生素对其有显著缓解作用（P〈0.05）;DES处理组仓鼠睾丸组织结构严重损伤,应用LBP后其损伤程度显著减轻,其中以10,50mg/kg LBP的作用最为明显,VC＋VE的缓解作用与50mg/kg LBP组接近;随着LBP剂量的增加,血清中SOD、GSH-Px的水平增加（P〈0.01）,而MDA的水平下降（P〈0.01）,维生素组与LBP组的变化趋势一致;LBP对血清激素水平也有不同程度的提高,其中以50mg/kg作用最为显著（P〈0.01）。这表明50mg/kg LBP能显著缓解DES致成年雄性仓鼠睾丸生精损伤。     

用微小隐孢子虫子孢子表面蛋白DNA疫苗免疫小鼠的实验研究

为了探讨微小隐孢子虫子孢子表面蛋白 CP15 / 60重组质粒 pc DNA3 .1- 15 / 60 DNA疫苗诱导机体产生体液和细胞免疫应答的效果。用重组的 DNA疫苗于 BAL B/ c小鼠后腿胫骨前肌肉注射免疫 ,于 0、3、6周共免疫 3次 ,10 0 μg/次。免疫后不同时间检测体液和细胞免疫应答指标。并用 1× 10 6 卵囊进行攻虫试验。结果表明 pc DNA 3 .1- 15 / 60可诱导机体产生相应的特异性抗体 ,对 C.parvum卵囊攻击具有保护作用。微小隐孢子虫子孢子表面蛋白 CP15 / 60重组质粒 pc DNA3 .1- 15 / 60有可能作为侯选的隐孢子虫 DNA疫苗 ,值得进一步深入研究     

犬瘟热病毒中国分离株囊膜糖蛋白基因免疫小鼠诱发的抗体应答

将CDV中国分离株(YZ0101)的两囊膜糖蛋白基因F和H与pGEM—rreasy载体构建的重组质粒分别采用EcoRI和Kprd、BamHI和Kpnl双酶切后定向克隆进真核表达载体pcDNA3．1(-)中，DNA测序和限制性酶切分析筛选阳性克隆pcDNA-H和pcDNA-F，以重组质粒DNA和脂质体共转染COS-7细胞，用间接免疫荧光试验验证转染的COS-7细胞胞浆中分别表达了CDV的H和F蛋白。以聚乙烯胺(PEI)为佐剂，将犬瘟热病毒F和H基因重组质粒pcDNA-H和pcDNA-F的DNA分别或混合肌注6周龄BABI／c小鼠，同时设pcDNA原载体DNA对照。以2周为间隔共免疫4次。最后一次免疫后3周，采血分离血清，酶联免疫吸附试验(ELISA)和病毒中和试验(SN)分析基因免疫在小鼠体内诱导的免疫应答。结果显示：pcDNA-H和pcDNA-F试验组免疫4次后分别激发了10^2.99 0.134、10^2.93 0.164滴度的ELISA抗体；两种质粒DNA混合免疫后产生了1：32～1：128的抗CDV的中和抗体；而原载体DNA免疫后，用ELISA和SN未检测出特异的CDV抗体。表明犬瘟热病毒囊膜糖蛋白F和H基因免疫小鼠可诱发产生特异性的体液免疫。     

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.