脂联素对酮病奶牛中性粒细胞自噬相关基因mRNA表达的影响

通过模拟向高BHBA的中性粒细胞培养液中添加不同质量浓度的脂联素(ADPN),运用荧光定量PCR技术,分别检测中性粒细胞自噬相关基因mRNA的表达水平。结果显示:添加高BHBA(4.8mol/L)的中性粒细胞培养6h后,与对照组相比,自噬相关基因LC3B、Beclin、ULK1的mRNA表达水平显著升高(P<0.01),SQSTM1(p62)、mTOR的mRNA表达水平显著下降(P<0.01);添加不同质量浓度的ADPN(25,50,100μg/L)后,相对于高BHBA组,中性粒细胞自噬相关基因LC3B、Beclin、ULK1的mRNA表达水平显著下调,且在ADPN 50μg/L时,具有极显著差异性(P<0.01);此外SQSTM1、mTOR的mRNA表达水平显著升高。结果表明:BHBA可增加中性粒细胞自噬相关基因的表达,ADPN可抑制高BHBA引起的中性粒细胞细胞程序性死亡,进一步说明ADPN对调控酮病奶牛中性粒细胞免疫抑制具有重要的价值意义。     

蓖麻蛋白对人外周血B淋巴细胞的作用及相关基因表达的研究

试验旨在研究蓖麻蛋白对人外周血B淋巴细胞（IM-9）毒性作用及对相关基因表达的影响。将提取的蓖麻蛋白按不同浓度添加到培养基培养6、12 h,研究剂量－时间效应,确定半数抑制浓度,然后以半数抑制浓度培养细胞2、6、10、12 h,在每个时间段检测免疫相关基因CD40、IL-1β、TNF-α和凋亡相关基因Bcl-2、Bax、Caspase-3的表达情况。结果表明,蓖麻蛋白对IM-9细胞的毒性作用随浓度和时间的增加而增强。取接近半数抑制浓度20 ng/mL作用IM-9细胞2、6、10、12 h,免疫相关基因中,TNF-α和CD40基因在6 h试验组表达量与对照组相比显著升高（P<0.05）,10、12 h达到极显著水平（P<0.01）;IL-1β基因表达量试验组、对照组间差异不显著（P>0.05）。凋亡基因中,与对照比相比,试验组Bax基因表达量10 h极显著降低（P<0.01）,12 h显著降低（P<0.05）;Bcl-2基因表达量4个时间段均达到显著水平（P<0.05）,2、12 h达到极显著水平（P<0.01）;Caspase-3基因除6 h表达量降低外,其他时间段表达量均显著升高（P<0.05）,2 h为极显著水平（P<0.01）。表明蓖麻蛋白能显著影响IM-9细胞的基因表达,TNF-α和CD40基因是两个潜在用IM-9细胞评价蛋白毒性的检测基因。     

山羊重组IL-17A克隆表达与生物活性分析

为了分析山羊白介素17A(IL-17A)对山羊单核、中性粒细胞功能的影响,采用RT-PCR方法对山羊IL-17A基因进行克隆,并在体外表达得到重组蛋白质(rIL-17A),以不同质量浓度的重组蛋白rIL-17A(5、10、20、40μg/mL)与单核细胞和中性粒细胞共孵育,测定rIL-17A对山羊单核细胞和中性粒细胞的迁移、中性粒细胞凋亡的免疫调节效应。结果显示:5、20、40μg/mL的rIL-17A能够极显著促进山羊单核细胞的迁移(P0.01),10μg/mL的rIL-17A也能显著促进山羊单核细胞的迁移(P0.05);与中性粒细胞共孵育时,10μg/mL(P0.05)和20、40μg/mL(P0.001)的rIL-17A能显著或极显著促进山羊中性粒细胞的迁移;20μg/mL(P0.05)、40μg/mL(P0.001)的rIL-17A能显著或极显著诱导山羊中性粒细胞的凋亡。结果表明:重组蛋白rIL-17A具有良好的生物学活性,能在体外不同程度地促进山羊单核细胞、中性粒细胞的迁移及中性粒细胞的凋亡。     

花生四烯酸对日本沼虾肝胰腺细胞脂质代谢基因表达的影响

本试验旨在评价细胞培养液中花生四烯酸(arachidonic acid,ARA)浓度对日本沼虾肝胰腺细胞活力及脂质代谢相关基因表达的影响。分离日本沼虾肝胰腺细胞,使用M199完全培养液培养5 d后换成含ARA的培养液,ARA浓度分别为0(ARA1)、50(ARA2)、100(ARA3)、200(ARA4)和1 000μmol/L(ARA5),测定12和24 h时脂质代谢相关基因的表达水平,以及24h时细胞活力。结果表明:原代肝胰腺细胞使用完全培养液时,生长状况良好,能存活15 d左右;ARA5组24 h时细胞活力显著低于ARA1和ARA2组(P0.05);高浓度的ARA降低了12和24 h时Δ4脱饱和酶(Δ4 FAD)、Δ6脱饱和酶(Δ6 FAD)、碳链延长酶6(Elovl6)、B类Ⅰ型清道夫受体(SR-BⅠ)、脂肪酸结合蛋白10(FABP10)、乙酰辅酶A结合蛋白(ACBP)基因表达水平;ARA作用12 h时,ARA2组SR-BⅠ基因表达水平显著高于其余各组(P0.05),ARA2和ARA3组FABP10基因表达水平显著高于ARA1和ARA5组(P0.05),ARA3组ACBP基因表达水平显著高于其余各组(P0.05);ARA作用24 h时,ARA2组SR-BⅠ、FABP10和ACBP基因表达水平显著高于其余各组(P0.05)。由此可见,细胞培养液中ARA浓度会影响日本沼虾肝胰腺细胞活力及脂质代谢相关基因的表达,过高的ARA浓度(1 000μmol/L)会降低细胞的活力,适宜的ARA浓度(50~100μmol/L)可促进脂肪酸脱饱和酶、碳链延长酶及脂肪酸转运相关基因的表达。     

脂多糖对大鼠乳腺上皮细胞酪蛋白基因表达的影响

本试验旨在探讨脂多糖(LPS)对大鼠乳腺上皮细胞中αs1-酪蛋白(CSN1S1)、β-酪蛋白(CSN2)和乳清蛋白(α-LA)3种主要酪蛋白基因表达的影响。采用单因素试验设计,用实时荧光定量PCR(RT-PCR)法研究在催乳素诱导作用下,0、100、500、1 000、5 000和25 000 ng/mL的LPS作用于HC11细胞3和24 h对酪蛋白基因(CSN1S1、CSN2和α-LA)、葡萄糖转运因子1(GLUT1)、葡萄糖转运因子8(GLUT8)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)基因表达的影响。结果表明:不同剂量、不同作用时间的LPS作用于HC11细胞,均显著抑制了CSN2基因的表达;作用3 h时,500～1 000 ng/mL的LPS显著增加了α-LA基因的表达量(P0.05),作用24 h时,所有剂量的LPS均显著抑制了α-LA基因的表达(P0.05);500～25 000 ng/mL的LPS作用3 h时和所有剂量的LPS作用24 h时均显著增加了CSN1S1基因的表达量(P0.05);LPS显著增加了TNF-α基因的表达量(P0.05);1 000 ng/mL的LPS作用3 h时和100～25 000 ng/mL的LPS作用24 h时,均显著增加了IL-6基因的表达量(P0.05);500～25 000 ng/mL的LPS作用3 h时显著增加了IL-1β基因的表达量(P0.05);从时间效应来看,总体趋势是3种细胞因子(IL-6、IL-1β、TNF-α)在作用24 h时的基因表达量均高于作用3 h时。大于5 000 ng/mL的LPS作用3 h时显著增加了GLUT1基因的表达量(P 0.05),所有剂量LPS均显著抑制了GLUT8基因的表达(P0.05)。综上,在大鼠乳腺上皮细胞中,LPS对不同酪蛋白基因的表达有不同的影响,LPS能抑制CSN2和α-LA基因的表达,却能增强CSN1S1基因的表达。     

波形蛋白对H9N2亚型AIV复制的影响研究

为研究波形蛋白(vimentin)对H9N2亚型禽流感病毒(AIV)复制的影响,构建了真核表达载体FLAG-vimentin,并采用Western blot技术验证FLAG-vimentin重组载体和病毒NP蛋白的表达情况,用荧光定量PCR技术检测病毒HA基因水平。同时,设计合成siRNA,敲低内源性vimentin,检测其对病毒HA基因表达水平的影响。构建原核表达载pCold I-vimentin,蛋白纯化后孵育细胞,采用Western blot、荧光定量PCR技术检测病毒的蛋白表达和基因水平的变化。结果证实,H9N2亚型AIV感染FLAG-vimentin重组载体转染的Hela细胞12 h时,病毒NP蛋白表达减少;而在感染24和36 h时,病毒NP蛋白表达上升。荧光定量PCR结果亦显示,在AIV感染后12 h时,HA基因表达水平明显降低。siRNA敲低Hela细胞内源性vimentin后,H9N2亚型AIV的HA基因表达水平在病毒感染12 h时明显升高,24 h时差异较小,36 h时有所升高。纯化后的vimentin重组蛋白孵育细胞,在病毒感染36 h时,H9N2亚型AIV的NP蛋...     

多不饱和脂肪酸对脂肪代谢酶基因表达的影响

多不饱和脂肪酸不仅可以抑制脂肪合成酶基因的表达,还能对脂质氧化相关酶基因的表达起促进作用,从而调节机体脂质代谢过程。本文主要就多不饱和脂肪酸的分类和来源、对脂肪合成及氧化相关酶基因表达的影响作一综述。     

白头翁汤及其拆方对cAMP-磷酸二酯酶活性影响的研究

通过检测白头翁汤水提物及其组方的各单味药对中性粒细胞c AMP-磷酸二酯酶(PDE)活性的影响,探讨其抗炎机理。结果表明白头翁汤较其组方的各单味药对中性粒细胞c AMP-PDE的抑制作用更强;白头翁汤组方中各单味药对中性粒细胞c AMP-PDE活性抑制作用是以黄连为主。该研究显示白头翁汤水提物可能是通过抑制c AMP-PDE活性,升高炎症细胞c AMP水平发挥抗炎作用。其抗炎成分及机理有待于进一步研究。     

乙酸浓度对奶牛乳腺上皮细胞甘油三酯含量及瘦素和过氧化物酶增殖物激活受体γ基因表达量的影响

本试验旨在研究乙酸浓度对奶牛乳腺上皮细胞甘油三酯(TAG)含量及瘦素(leptin)和过氧化物酶增殖物激活受体γ(PPARγ)基因表达量的影响。将传至第3代的奶牛乳腺上皮细胞以适宜的密度培养48 h后,随机分为6个处理,各处理分别采用乙酸浓度为0(对照组)、4、6、8、10和12 mmol/L的培养液。置于37℃5%CO2培养箱继续培养48 h。收集细胞,观测脂滴形成状况,测定TAG含量及leptin和PPARγ基因表达量。结果表明:随乙酸浓度的增加,培养的奶牛脂肪前体细胞脂滴形成增多;随乙酸浓度的增加,TAG含量和PPARγ基因表达量均呈显著的线性或二次曲线增加(P<0.05),且以乙酸浓度为10和12 mmol/L时效果较好;一定浓度的乙酸可上调leptin基因表达量,尤以浓度为8、10 mmol/L时上调作用较好。结果提示,乙酸对奶牛乳腺上皮细胞内脂滴的形成、TAG的积累、leptin和PPARγ基因的表达有显著的促进作用。本试验条件下,乙酸浓度为10～12 mmol/L时能较好地促进PPARγ基因的表达,浓度为8～10 mmol/L时能较好地促进leptin基因的表达。     

鸡前脂肪细胞中A-FABP基因表达的变化对PPARγ、perilipin和E-FABP表达的影响

本研究以鸡原代前脂肪细胞为实验材料,探讨A-FABP基因在鸡脂类代谢过程中的功能以及该基因与脂类代谢相关7个基因间的调控关系.利用A-FABP基因的shRNA干扰载体和真核表达载体对该基因进行干扰和过表达,分别在干扰和过表达后24、36、48、60和72 h时检测脂类代谢相关基因的表达情况.结果表明,干扰A-FABP后,鸡前脂肪细胞中PPARγ和perilipin基因的表达量在24和36 h时显著低于对照组,在48 h时显著高于对照组,在48和72 h时,E-FABP基因表达量显著低于对照组；过表达A-FABP后,鸡前脂肪细胞中PPARγ和perilipin基因的表达量在24和36 h时显著高于对照组,在48 h时显著低于对照组,在所有检测的时间点E-FABP基因表达均显著下调.结果显示,在前脂肪细胞中,鸡A-FABP表达量的变化影响PPARγ、perilipin和E-FABP的表达.     

Characterization of arginase expression by equine neutrophils

Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood neutrophils using quantitative PCR. Arginase expression was also studied by Western blot and enzyme activity assay. The effect of nor-NOHA (1 mM), a specific arginase inhibitor, was assessed on arginase activity in vitro and ex vivo on neutrophil's inflammatory gene expression and viability. Results showed that equine neutrophils constitutively express arginase isoform 2, ODC and OAT. Neutrophil ex vivo stimulation did not induce arginase I or influence arginase II mRNA expression. Ex vivo inhibition of arginase activity by nor-NOHA had no effect on neutrophils inflammatory gene expression induced by LPS/fMLP (5 h) but significantly reversed the cell loss observed after this stimulation.     

磷酸二酯酶在猪卵巢的表达与相关中药对其活性的影响

为了测定猪卵巢组织磷酸二酯酶（PDE）同工酶基因表达类型与活性规律，研究相关中药的作用机理，为繁殖障碍性疾病治疗积累资料。作者提取中国实验用小型猪卵巢组织总RNA，应用反转录聚合酶链反应（RT-PCR）测定PDE同工酶在其中的表达。选取催情散等复方及单味药，运用高效液相色谱法（HPLC）根据作用前后cAMP/cGMP含量的变化，分析对PDE活性的影响。结果检测的18个PDE亚型中除PDE4C外，PDE 1A、1B、1C、2A、3A、3B、4A、4B、4D、5A、7A、7B、8A、8B、9A、10A、11A mRNA在卵巢组织中均有表达。卵巢组织PDE活性较强，其中cGMP-PDE活性大于cAMP-PDE活性。催情散对卵巢组织cGMP-PDE活性均有较强的抑制作用，其中单味药淫阳藿作用最强。研究结果表明卵巢组织含有丰富的PDE同工酶，在通过环核苷酸水平卵巢机能调节中发挥着重要作用，中药催情散的作用与显著抑制cGMP-PDE活性有关。     

磷酸二酯酶基因在小型猪骨骼肌组织的表达

提取中国实验用小型猪骨骼肌组织总RNA，应用反转录聚合酶链反应测定磷酸二酯酶(PDE)同工酶在骨骼肌组织中的表达分布。结果可见PDE1A、1C、2A、3A、3B、4A、4B、4C、4D、5A、7A、7B、8A、8B、9A和11A共16种PDE同工酶mRNA在中国实验用小型猪骨骼肌组织中表达，其中PDE1A、1C、2A、3A、4B、4C、8A和8B共8种在人和其他动物骨骼肌组织中的表达分布未见报道，PDE1B和10A3种同工酶未见表达。16种PDE同工酶mRNA在骨骼肌组织中的表达，从电泳条带上可以看到各种PDE表达量存在着差异，但需要进一步做定量分析加以评定。     

猪磷酸二酯酶4B2的原核表达和活性鉴定

使用RT-PCR方法扩增猪磷酸二酯酶4B2基因,将其克隆到表达载体,经PCR和测序鉴定的阳性重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白表达。结果显示,目的基因片段大小为1718 bp,与GenBank公布的猪PDE4B2基因序列同源性为99.7%。表达的重组蛋白以包涵体和可溶性蛋白两种形式存在。Western blotting检测结果表明,蛋白质大小约为66 ku,通过液相检测重组蛋白的cAMP-PDE活性为51.46%。制备的多克隆抗体有良好的特异性,为进一步检测天然蛋白PDE4B2和筛选PDE4B2的特异性抑制剂奠定了基础。     

采食后奶牛瘤胃短链脂肪酸及其吸收相关蛋白表达的研究

本试验旨在研究奶牛采食前后瘤胃中短链脂肪酸(SCFA)浓度的变化及其吸收相关蛋白表达量的差异。试验选用3头体重(720±30)kg且装有瘘管的健康荷斯坦牛(动物伦理审查编号为SXAU-EAW-2019-C002013),采食精粗比为40:60的日粮(10kg),试验预试期10d,于第11天饲喂前开始取样,采用气相色谱法检测奶牛采食前(0 h)和采食后(1、2、3、4、5、6、7、8 h)瘤胃液中SCFA浓度;并采用荧光定量PCR方法检测瘤胃上皮组织中与SCFA吸收相关的蛋白表达量。结果表明:在采食后1 h奶牛瘤胃中SCFA浓度最高(P<0.05);在采食后一段时间内(2~5h)与SCFA吸收相关蛋白表达量上调(P<0.05),AE2、MCT1基因表达量均在5 h最高,PAT1、NHE3基因表达量均在4 h最高,MCT4基因表达量在4、5、6h均较高,NHE1基因表达量在2h达到最高;AE2、MCT1、MCT4、NHE1基因表达量与SCFA浓度负相关或正相关(P<0.05),AE2、MCT1、MCT4基因表达量与瘤胃内pH正相关(P<0.05)。以上结果初步揭示,在采食后一定时间内,瘤胃中与SCFA吸收相关蛋白表达受SCFA浓度和pH的调节。     

Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle.

L-selectin (CD62L) gene expression in neutrophils is commonly referred to as "constitutive" because circulating neutrophils require a constant supply of this adhesion molecule for continuous trafficking into peripheral tissues. Under normal circumstances, marginating blood neutrophils and neutrophils that become activated for migration into infected tissues rapidly shed surface CD62L that is ligated to the vascular endothelium. However, this does not shut down CD62L gene expression because these cells continue to express surface CD62L. In contrast, glucocorticoid challenges resulting from stress and hormone injections result in gradual and chronic down-regulation of CD62L on the surface of blood neutrophils. Rather than being associated with migration, this type of CD62L down-regulation associates with pronounced neutrophilia and increased susceptibility to infections. Nothing is currently known about glucocorticoid regulation of CD62L expression in neutrophils. In other cell systems, however, this steroid hormone binds to cytoplasmic glucocorticoid receptors (GR) that influence expression of glucocorticoid-responsive genes at multiple pre-translational levels. Thus, the hypothesis of the present study was that glucocorticoid challenge suppresses CD62L mRNA expression in blood neutrophils. Suppressed CD62L gene expression might help explain the chronic down-regulation of surface CD62L in neutrophils and accompanying neutrophilia. The main objectives of the study were to monitor neutrophil CD62L mRNA abundance before and during subtle and severe glucocorticoid challenges and to determine if CD62L mRNA expression correlates with degree of glucocorticoid challenge. Parturient dairy cows and dexamethasone-treated steers were used as models of subtle and severe (respectively) glucocorticoid challenges. Data presented from both models support the hypothesis and show for the first time that glucocorticoids regulate neutrophil CD62L at a pre-translational level. Results also showed that inhibited CD62L mRNA expression correlated precisely with down-regulated surface expression of CD62L on neutrophils and peak neutrophilia during severe glucocorticoid challenge. Therefore, results of this study indicate that bovine neutrophils are highly sensitive to the blood environment, displaying full capacity to alter CD62L gene expression and trafficking patterns in response to changing glucocorticoid levels. This may serve animals well when heightened inflammatory responses begin to lead to tissue damage, but may be detrimental to overall health if animals are exposed to opportunistic pathogens while stressed or undergoing glucocorticoid therapy. Although this study did not elucidate how glucocorticoids inhibit neutrophil CD62L mRNA expression, presented data implicate GR as possibly being involved because neutrophils from cattle in both models expressed GR mRNA. Further in vitro studies using purified populations of neutrophils will be required to determine if GR is directly involved in glucocorticoid regulation of CD62L gene expression and, if so, at what level.     

Innate immune responses of temperamental and calm cattle after transportation

The objective was to investigate measures of cellular innate immune responses among calm and temperamental Brahman bulls in response to handling and transportation. Sixteen Brahman bulls (344 ± 37 d of age; 271.6 ± 45.5 kg BW) classified as either calm (n=8) or temperamental (n=8) were loaded onto a trailer, transported for 4h to a novel facility, rested 16 h overnight, and then were returned to their original facility after a 4h transport. Blood samples were collected immediately prior to (time 0) and at 24, 48, and 96 h after initial loading for analyses of innate immune and blood parameters. Leukocyte counts did not differ (P>0.05) due to temperament before or after transportation, but neutrophil:mononuclear cell ratios were greater in temperamental bulls compared to calm bulls at 24h. At 24h, expression of peripheral neutrophil β(2)-integrin decreased among all bulls compared with 0 h (P<0.01). Temperamental bulls had greater glucose and cortisol than calm bulls (P<0.01) at 48 h; whereas calm bulls had elevated neutrophil L-selectin expression, and phagocytic and oxidative burst activity compared with temperamental bulls (P<0.10) at 48 h. The supernatant collected from endotoxin-stimulated whole blood cultures had greater TNF-α concentrations at 48 h than at the other time points (P<0.05), but no temperament effect was observed (P>0.05). In contrast, 96 h after initial loading the supernatant TNF-α concentrations were lower (P<0.05) among all cattle. Lastly, transportation increased neutrophil phagocytosis, oxidative burst, and cell adhesion molecule expression 96 h post-transportation and the effect was more pronounced among calm bulls. These data suggest that neutrophils from calm bulls are more likely to resist microbial invasion at 96 h after transportation than neutrophils from temperamental bulls.     

Effect of prostaglandin F2 alpha on the adenylate cyclase and phosphodiesterase activity of ovine corpora lutea

The objective of the present study was to determine the temporal relationships among luteal adenylate cyclase activity, luteal phosphodiesterase activity, luteal progesterone concentration and plasma progesterone concentration during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis in ewes. Corpora lutea (CL) were removed from cycling ewes on d 9 (d 0 = first day of estrus) at 0, 2, 4, 6, 12 and 24 h (seven to eight ewes/group) after PGF2 alpha administration (im). Jugular blood samples were collected at the time of enucleation of CL and analyzed for progesterone. Plasma and luteal progesterone concentrations were decreased (P less than .05) by 4 and 12 h after PGF2 alpha injection, respectively. Basal adenylate cyclase, luteinizing hormone (LH)-activated adenylate cyclase, guanylylimidodiphosphate [Gpp(NH)p]-activated adenylate cyclase and LH plus Gpp(NH)p-activated adenylate cyclase activities were decreased (P less than .05) by 2 h after PGF2 alpha injection. The decrease in adenylate cyclase activity paralleled the decrease in plasma progesterone concentration over time. Luteinizing hormone stimulated (P less than .05) adenylate cyclase activity relative to basal activity at 0, 2, 12 and 24 h post-PGF2 alpha; whereas, Gpp(NH)p stimulated (P less than .01) adenylate cyclase activity relative to basal activity at each time point. In contrast to the decrease in adenylate cyclase activity, phosphodiesterase activity was increased (P less than .05) at 2 and 4 h post-PGF2 alpha. These results suggest that a decrease in adenylate cyclase activity coupled with an increase in phosphodiesterase activity may decrease the intracellular adenosine 3',5' cyclic monophosphate (cAMP) concentration.(ABSTRACT TRUNCATED AT 250 WORDS)