化脓隐秘杆菌溶血素和铁结合蛋白的抗体应答分析

目的 对化脓隐秘杆菌（Trueperella pyogenes）溶血素（pyolysin,PLO）和铁结合蛋白（iron-binding protein,IBP）的抗体应答情况进行分析。方法 采用腹腔注射途径建立化脓隐秘杆菌小鼠感染模型,观察不同剂量细菌对小鼠的致死性和注射部位形成的组织病变。制备化脓隐秘杆菌灭活抗原,采用肌肉注射途径免疫45只雌性昆明小鼠和10只山羊,每只小鼠0.2 mL,每只山羊1 mL,均进行3次免疫,每次间隔2周,第3次免疫后2周采血分离血清。采用ELISA法测定感染小鼠、健康圈养羔羊、化脓隐秘杆菌灭活抗原免疫小鼠和山羊血清中抗PLO和IBP的IgG抗体水平。结果 注射部位出现化脓和皮肤坏死的小鼠血清中能检出高水平的抗PLO和IBP抗体;无组织病变小鼠血清中仅能检测到低水平的抗IBP抗体;连续注射无组织病变剂量细菌的小鼠血清能检出抗PLO和IBP抗体。圈养羔羊血清抗PLO抗体阳性率高,而抗体水平低。免疫小鼠和山羊血清中均能检出抗IBP抗体,但部分动物检测不出抗PLO抗体。结论 IBP是化脓隐秘杆菌感染和灭活疫苗免疫诱导抗体应答的主要抗原之一,PLO抗体水平具有反...     

山羊化脓隐秘杆菌溶血素抗体间接ELISA检测方法的建立、验证及应用

目的 建立山羊化脓隐秘杆菌溶血素(pyolysin,PLO)抗体的间接ELISA检测方法,并进行验证及应用.方法 通过E coli表达系统表达重组溶血素(recombinant pyolysin,rPLO),采用Ni-NTA亲和层析进行纯化.以rPLO作为包被抗原,HRP标记的兔抗山羊IgG抗体为二抗,建立检测山羊化脓...     

黄芩苷通过Bcl-2/Caspase-3信号通路促HepG2细胞凋亡

目的：探究黄芩苷对人肝癌细胞HepG2是否具有促凋亡效应及其相关作用机制。方法：通过MTT法检测了黄芩苷对HepG2的增殖抑制效应;流式细胞分析检测了黄芩苷对HepG2凋亡的影响;Western Blot实验和q-RTPCR实验检测了黄芩苷对细胞凋亡相关分子Caspase-3、Bcl-2、Bax的表达影响。结果：黄芩苷对人肝癌细胞HepG2存在明显的增殖抑制效应,诱导细胞发生了凋亡,且引起Caspase-3与Bax表达上调,Bcl-2表达下调。结论：黄芩苷通过Bcl-2/Caspase-3信号通路促HepG2细胞发生凋亡,为中药药物抗肿瘤机制研究提供参考。     

白藜芦醇基于PI3K/Akt通路对人结肠癌细胞HCT-116凋亡的影响

目的：探究白藜芦醇基于PI3K/Akt通路对人结肠癌细胞HCT-116凋亡的影响及其相关分子机制。方法：通过MTT实验检测白藜芦醇对HCT-116细胞的增殖抑制效应;流式细胞仪检测白藜芦醇对HCT-116的促凋亡效应;蛋白免疫印迹实验检测HCT-116细胞中Caspase-3、Bcl-2、Bax、PI3K、Akt表达情况的影响。结果：白藜芦醇对HCT-116有明显的抑制增殖作用,并有效促进了其凋亡,且引起Caspase-3与Bax表达上调,Bcl-2表达下调并抑制了PI3K和Akt的磷酸化。结论：白藜芦醇可诱导HCT-116细胞发生凋亡,且与抑制PI3K/Akt信号通路的激活相关。     

秦巴硒菇提取物FA-2-b-β对伯基特淋巴瘤Raji细胞增殖及凋亡的影响及其作用机制

目的观察秦巴硒菇提取物酸性RNA蛋白复合物FA-2-b-β对伯基特淋巴瘤细胞株Raji增殖及凋亡的影响,并探讨其相关作用机制。方法体外培养Raji细胞,分别加入浓度为0.9、1.5、2.1、2.7 mg/mL的FA-2-b-β,对照组加入等量培养基。培养24、48、72 h后收集各组细胞,采用CCK-8法检测FA-2-b-β对Raji细胞的增殖抑制作用;流式细胞仪检测细胞凋亡率;Western blot法检测细胞β-catenin、c-myc、cyclin D1、Bcl-2、Bax蛋白的表达。结果 FA-2-b-β能抑制Raji细胞增殖,且呈浓度-时间依赖性(P <0.01);给药48 h后,各浓度FA-2-b-β组细胞凋亡率较对照组均显著上升,且呈剂量依赖性(P <0.05);各浓度FA-2-b-β组细胞β-catenin、c-myc、cyclin D1、Bcl-2蛋白的表达水平显著低于对照组,且呈剂量依赖性(P <0.05),Bax蛋白表达水平显著高于对照组,同样呈剂量依赖性(P <0.05),Bax/Bcl-2比值增加。结论 FA-2-b-β呈时间-剂量依...     

氟离子对氧化硫硫杆菌的影响

对细菌HSS用于生物浸出磷矿进行了研究.研究发现,磷矿中所含的氟离子对细菌活性起决定性影响.随着培养液中氟离子浓度的增大,细菌的生长和产酸滞后期变长,其生长量和氧化硫磺的能力也大为降低.细菌的耐氟能力可以通过驯化方式提高,通过浸矿筛选优良菌种能加快细菌的耐氟驯化速度.     

牛坏死杆菌43K OMP单克隆抗体的制备及鉴定

目的 制备牛坏死杆菌43KOMP单克隆抗体,并鉴定其生物学特性.方法 经IPTG诱导表达重组43KOMP,纯化后免疫BALB/c小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选分泌抗43K OMP特异性抗体的杂交瘤细胞,制备腹水,对抗43K OMP单克隆抗体进行效价、亚类、免疫原性及特异性鉴定.结果 获得3株稳定表达抗43...     

姜黄素对HeLa细胞增殖和凋亡的影响

目的探讨姜黄素对HeLa细胞增殖和凋亡的影响及其机制。方法分别用10、20、40μmol/L姜黄素处理HeLa细胞24h后,MTT法检测细胞的增殖,光镜观察细胞形态,TUNEL技术检测细胞的凋亡,免疫细胞化学法检测Cytochrome C和Caspase-9蛋白的表达,Western blot法检测XIAP蛋白的表达。结果姜黄素对HeLa细胞的增殖有抑制作用,并呈剂量依赖性;部分细胞呈现典型的凋亡形态学改变;姜黄素作用后,Cytochrome C和Caspase-9蛋白的表达显著增强,XIAP蛋白的表达显著下降,且均呈剂量依赖性。结论姜黄素能抑制HeLa细胞增殖并诱导其凋亡,Cytochrome C和Caspase-9的表达上调及XIAP的表达下调可能参与凋亡过程。     

氟离子对氧化硫硫杆菌的影响

对细菌HSS用于生物浸出磷矿进行了研究,研究发现,磷矿中所含的氟离子对细菌活性起决定性影响,随着培养液中氟离子浓度的增大,细菌的生产和产酸滞后期变长,其生长量和氧化硫磺的能力也大的降低,细菌的耐氟能力可以通过驯化方式提高,通过浸矿筛选优良菌种能加快细菌的耐氟驯化速度。     

底物对亚铁硫杆菌生物氧化过程的影响

实验表明T .ferrooxidans对FeSO4和硫等单或双底物利用的难易程度次序为 :单底物培养基中FeSO4最易利用 ,Na2 S2 O3 其次 ,单质硫最难利用 ;双底物培养基中 ,T .Ferrooxidans首先利用Fe2 + ,然后利用硫 ,此外培养液中还伴随发生化学氧化反应、中和沉淀、生物氧化等反应 ,以上反应使培养液中亚铁的浓度在培养中期上下波动直至铁氧化酶抑制被解除。     

Effects of Luteolin on Biofilm of Trueperella pyogenes and Its Therapeutic Effect on Rat Endometritis

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals. The development of new anti-biofilm drugs will improve the current treatment status for controlling T. pyogenes infections in the animal husbandry industry. Luteolin is a naturally derived flavonoid compound with antibacterial properties. In this study, the effects and the mechanism of luteolin on T. pyogenes biofilm were analyzed and explored. The MBIC and MBEC of luteolin on T. pyogenes were 156 μg/mL and 312 μg/mL, respectively. The anti-biofilm effects of luteolin were also observed by a confocal laser microscope and scanning electron microscope. The results indicated that 312 μg/mL of luteolin could disperse large pieces of biofilm into small clusters after 8 h of treatment. According to the real-time quantitative PCR detection results, luteolin could significantly inhibit the relative expression of the biofilm-associated genes luxS, plo, rbsB and lsrB. In addition, the in vivo anti-biofilm activity of luteolin against T. pyogenes was studied using a rat endometritis model established by glacial acetic acid stimulation and T. pyogenes intrauterine infusion. Our study showed that luteolin could significantly reduce the symptoms of rat endometritis. These data may provide new opinions on the clinical treatment of luteolin and other flavonoid compounds on T. pyogenes biofilm-associated infections.     

Effect of abomasal infusions of geometric isomers of 10,12 conjugated linoleic acid on milk fat synthesis in dairy cows

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) decreases TAG accumulation in 3T3-L1 adipocytes, reduces lipid accretion in growing animals, and inhibits milk fat synthesis in lactating mammals. However, there is evidence to suggest that other FA may also exert antilipogenic effects. In the current experiment, the effects of geometric isomers of 10,12 CLA on milk fat synthesis were examined using four Holstein-British Friesian cows in a 4×4 Latin Square experiment with 14-d periods. Treatments consisted of abomasal infusions of skim milk, or skim milk containing trans-10, cis-12 CLA (T1), trans-10, trans-12 CLA (T2), or a mixture of predominantly 10,12 isomers containing (g/100 g) trans-10, cis-12 (35.0), cis-10, trans-12 (23.2), trans-10, trans-12 (14.9), and cis-10, cis-12 (5.1). CLA supplements were prepared from purified ethyl linoleate and infused as nonesterified FA. Infusions were conducted over a 4-d period with a 10-d interval between treatments and targeted to deliver 4.5 g/d of 10,12 CLA isomers. Compared with the control, trans-10, trans-12 CLA had no effect (P>0.05) on milk fat yield, whereas treatments T1 and T3 depressed (P<0.05) milk fat content (19.8 and 22.9%, respectively) and decreased milk fat output (20.8 and 21.3%, respectively). Comparable reductions in milk fat synthesis to 4.14 and 1.80 g trans-10, cis-12/d supplied by treatments T1 and T3 indicate that other 10,12 geometric isomers of CLA have the potential to exert antilipogenic effects. The relative abundance of cis-10, trans-12 CLA in treatment T3 and the low transfer efficiency of this isomer into milk suggest that cis-10, trans-12 CLA was the active component.     

Suppressive effect of docosahexaenoyl‐lysophosphatidylcholine and 17‐hydroxydocosahexaenoyl‐lysophosphatidylcholine on levels of cytokines in spleen of mice treated with lipopolysaccharide

Lysophosphatidylcholine (lysoPC) with polyunsaturated fatty acyl chains has been known to be anti‐inflammatory in vivo. In the present study, we examined the effect of docosahexaenoyl‐lysophosphatidylcholine (DHE‐lysoPC) and 17‐hydroxydocosahexaenoyl‐lysophosphatidylcholine (17‐HDHE‐lysoPC) on spleen weight and cytokine level in spleen of mice treated with lipopolysaccharide (LPS). For this purpose, mice were administrated i.p. with DHE‐lysoPC or 17‐HDHE‐lysoPC 1 h before i.p. injection of LPS. First, DHE‐lysoPC (50–400 µg/kg) was found to suppress the LPS‐induced increase of spleen weight dose‐dependently, and such a suppressive effect was greater for 17‐HDHE‐lysoPC, compared to DHE‐lysoPC. Next, in an attempt to see the effect of DHE‐lysoPC on cytokine levels in spleen of mice treated with LPS, DHE‐lysoPC was found to suppress LPS‐induced increase in the levels of cytokines such as TNF‐α, IL‐1β, or IL‐6 in a dose dependent manner (50–400 µg/kg), in contrast to DHA showing a significant action at a high dose (400 µg/kg) only. The greater suppressive effect of 17‐HDHE‐lysoPC (15–150 µg/kg) than DHE‐lysoPC suggested that action of DHE‐lysoPC may be enhanced through lipoxygenation process. Presumably in support of this, when the interval time between 17‐HDHE‐lysoPC administration and LPS challenge was varied, the cytokine‐suppressing effect was found to be augmented in a time‐dependent manner. Taken all together, it is suggested that DHE‐lysoPC and 17‐HDHE‐lysoPC may be beneficial in suppressing the inflammation in spleen tissue.     

Effect of CLA on milk fat synthesis in dairy cows: Comparison of inhibition by methyl esters and free fatty acids,and relationships among studies

CLA is a potent inhibitor of milk fat synthesis, as shown by investigations using mixtures of CLA isomers in FFA form. However, methyl esters of CLA can be initially formed in commercial synthesis, and their use in a supplement has certain manufacturing and cost advantages. Our objective was to compare abomasal infusion of methyl esters of CLA (ME-CLA) and FFA of CLA (FFA-CLA) on milk fat synthesis. Data were also combined with previous investigations to examine broader relationships between trans-10,cis-12 CLA and the reduction in milk fat. Three mid-lactation, rumen-fistulated Holstein cows were used in a 3×3 Latin square design. Treatments were (i) control, (ii) ME-CLA, and (iii) FFA-CLA. The ME-CLA and FFA-CLA treatments (4.2 g/d trans-10,cis-12 CLA) resulted in a comparable reduction in milk fat yield (38 and 39%, respectively) and pattern of reduction in individual FA. In contrast, milk yield, milk protein, and feed intake were unaltered by CLA treatment. Combining data across studies revealed strong correlations relating the reduction in milk fat yield to abomasal dose of trans-10,cis-12 CLA (R ²=0.86), milk fat content of trans-10,cis-12 CLA (R ²=0.93), and milk fat secretion of trans-10,cis-12 CLA (R ²=0.82). Across studies, transfer efficiency of abomasally infused trans-10,cis-12 CLA into milk fat was relatively constant (22%; R ²=0.94). Overall, ME-CLA and FFA-CLA were equally potent in reducing milk fat, and either form could be used to formulate a dietary supplement that would induce milk fat depression.     

The influence of dietary rumen‐protected linoleic acid on milk fat composition,spreadability of butter and energy balance in dairy cows

The aim of this study was to determine the effects of a diet supplemented with rumenprotected linoleic acids (C18:2) on the composition of milk fat and the energy balance of dairy cattle during the first 15 wk of lactation. The 32 Holstein‐Friesian cows were allotted in two treatment groups; in the experimental group one‐third of the starch (relative to the control group) was substituted with protected fat on an energy basis. Milk samples from all cows were collected weekly from week 2 to 15 postpartum (p.p.). To analyze the milk fat composition milk samples from 16 cows in each group were collected from week 6 and 7 as well as from week 13 and 14 p.p. and were mixed together, respectively. Triglyceride analysis demonstrated an extensive use of depot fat in both cow groups at the beginning of the lactation period. However, calculated energy balance, triglyceride composition and back fat thickness showed that the usual deficit of energy intake in early lactation was significantly shortened in the experimental group by three weeks. In comparison with the control group the content of the saturated fatty acids (FAs) C12, C14 and C16 in the experimental group decreased by 17.3% at 6 to 7 wk and by 19.2% at 13 to 14 wk. The stearic acid content of milk fat was increased by 25.9% at 6 to 7 wk and by 27.7% at 13 to 14 wk in the experimental group. The content of cis Δ9 oleic acid was increased by 21.6% at 6 to 7 and by 30.3% at 13 to 14 wk, while the C18:2 FA content was doubled as compared with the control group. Thus besides the increase of the trans‐C18:1 FA (TFA) content the nutritional value of fats could be improved using the experimental fat supplement. The TFA content still remained within the range of variation of natural milk fats. Additionally the experimental fat intake led to a number of desired effects; an increase in the content of conjugated linoleic acids (cis Δ9, transΔ11) by 55.9% (6 to 7 wk) and by 97.1% (13 to 14 wk p.p.), respectively, and a decrease in the cholesterol level. Further, the butyric acid content increased relatively by more than 20%. The addition of this fat resulted simultaneously in a changed triglyceride composition with increased C50, C52 and C54 contents. Thus a markedly improved spreadability of the resulting butter might be expected.     

骨髓基质干细胞对大鼠肝纤维化细胞凋亡的影响

目的探讨骨髓基质干细胞(BMSCs)对大鼠肝纤维化细胞(CFSCs)凋亡的影响。方法常规培养BMSCs、CFSCs和大鼠肝细胞系BRL,并双层共培养CFSCs与BMSCs。用ELISA方法检测BMSCs培养上清中NGF、HGF和TGF-β1的浓度;RT-PCR检测与BMSCs共培养的CFSCs及BRL的p75表达;TUNEL法检测BMSCs分泌的细胞因子封闭后对CFSCs凋亡的影响。结果BMSCs可分泌NGF、HGF、TGF-β1等细胞因子,且随着培养时间的延长,其分泌量逐渐增多;BRL不表达p75,CFSCs表达p75,且与BMSCs共培养后,其表达量增高;分别用p75的阻断剂TAT-Pep5、HGF的中和抗体和JNK的阻滞剂sp600125作用后,BMSCs诱导CFSCs凋亡的比例均明显降低;封闭TGF-β1后,BMSCs诱导CFSCs凋亡的比例增高。结论BMSCs能够通过分泌NGF和HGF促进CFSCs的凋亡,这种凋亡诱导作用依赖于JNK的活性,并且在封闭TGF-β1后增强。