牛MEN1基因真核表达载体的构建及其表达分析

多发性内分泌腺瘤致病因子1 (multiple endocrine neoplasia Ⅰ,MEN1)参与乳腺发育与泌乳行为的调控.本研究克隆了牛(Bos taurus) MEN1基因(bMEN1)的全长cDNA,并在不同细胞中检测bMEN1mRNA及其编码蛋白menin的表达情况.根据GenBank中bMEN1基因序列,设计特异性引物,用qRT-PCR方法得到附带EcoR Ⅰ和HindⅢ酶切位点的bMEN1片段,并将其克隆到真核表达载体pcDNA3.1-myc-his(A)中.体外转染物种同源性细胞牛乳腺上皮细胞(bovine mammary gland epithelial cell,MAC-T)和异源性细胞中国仓鼠(Cricetulus barabensis)卵巢细胞(Chinese hamster ovary cells,CHO)、小鼠(Mus musculus)成肌细胞(mouse myoblast cells,C2C12),利用qRT-PCR和Westem blot技术检测转染前后bMEN1 mRNA和蛋白menin的表达.双酶切和基因测序结果表明,成功构建了bMEN1基因的真核表达载体pcDNA3.1-myc-his-bMEN1.所建立的转染体系可以使目的基因bMEN1在3种不同细胞中成功表达mRNA和目的蛋白,转染后24 h都达到最高表达量,并达到极显著水平(P＜0.01),之后逐渐降低.其中,CHO细胞中的转染24 h后bMEN1 mRNA和menin蛋白的表达量分别是对照的28 415倍和5.65倍.本研究建立了bMEN1基因的真核表达载体及其转染体系,能够在不同细胞中成功表达,为体外研究MEN1基因对于乳腺的调节功能及其对于机体代谢的调节机制提供了一种工具和技术体系.     

应用双抗体夹心法ELISA检测IBDV的研究

从抗ＩＢＤＶ高免蛋黄液中提取ＩｇＧ，用作包被抗体和酶标记，建立了检测ＩＢＤＶ的双抗体夹心法ＥＬＩＳＡ。经抗原阻断，无关病毒对照、取代、验证等项试验，并与常规ＡＧＰ法比较，对来源不同的１００多个样品进行检测，结果表明：该法对患鸡腔上囊、脾脏的检出率均为１００％，比ＡＧＰ法敏感１００倍以上，用肉眼观察阳性与阴性之间颜色差异显著，不存在非特异性反应。试验证明该法具有满意的待异性、敏感性、快速性和稳定性，是ＩＢＤ早期病原学诊断和流行病学调查的有效手段。     

Physiological and biochemical parameters for egg quality determination in lake trout,Salmo trutta lacustris

The present study investigated the relationships between egg viability and ovarian fluid composition, egg physiology and egg metabolism in lake trout, Salmo trutta lacustris, to obtain biomarkers for egg quality determination. The ovarian fluid pH, protein levels and activities of aspartate aminotransferase and -d-glucuronidase were significantly correlated with egg viability expressed as the number of eyed stage embryos. Regression models demonstrated that an ovarian fluid pH between 8.44 and 8.57, protein levels below 235.56 mg 100 ml^–1ovarian fluid, aspartate aminotransferase activity below 31.65 m min^–1 l^–1ovarian fluid and -d-glucuronidase activity below 8.62 m min^–1 l^–1 ovarian fluid characterized egg batches with high viability (80%).The increase in the egg wet weight during water hardening was also significantly correlated with the number of eyed stage embryos, and egg batches with high egg viability (80%) increased in wet weight by 13% during water hardening.From the investigated metabolic parameters the number of eyed stage embryos was significantly correlated with activities of NADP-dependent isocitrate dehydrogenase (egg viability 80% at 2.07 nM min^–1 mg^–1 protein) and NAD-dependent malate dehydrogenase (egg viability 80% at 47.25 nM min^–1 mg^–1 protein), with the respiration rate (egg viability 80% at 8.71 nM min^–1 mg^–1 protein), with the ratio of NADH to NAD levels (egg viability 80% 0.872), with the levels of free, non-esterified fatty acids (egg viability 80% 72.34 g mg^–1 protein), and the ratio of non esterified to esterified fatty acids (egg viability 80% at 0.749). Also, subjective and visual control methods were described to distinguish between batches with viable and non viable eggs.     

Wnt5a介导Wnt/Ca2+通路对BCG诱导牛原代肺泡上皮细胞自噬的调控作用

旨在探讨体外分离培养牛肺泡上皮细胞（bovine alveolar epithelial cells,BAECs）的方法及Wnt5a对牛结核分枝杆菌卡介苗（bacille Calmette-Guérin,BCG）感染BAECs细胞自噬的调控机制。试验选用酶联合消化法和机械刮刷法分离细胞,差速贴壁法纯化BAECs,免疫荧光染色检测上皮细胞标志物角蛋白14（cytokeratin 14,CK14）和角蛋白5（cytokeratin 5,CK5）的表达;BCG感染BAECs,并用Box-5抑制Wnt5a的表达,Western blot和免疫荧光染色检测自噬相关蛋白及非经典Wnt信号通路相关蛋白的表达。结果表明,采用酶联合消化法和机械刮刷法能够成功分离纯度较高的BAECs,细胞经CK14和CK5鉴定为阳性;BCG感染BAECs促进Wnt5a表达,增加细胞自噬,Box-5预处理下调BCG诱导的细胞自噬相关蛋白LC3II、P62、Atg7及Atg5的表达,且抑制非经典Wnt/Ca²⁺信号通路相关蛋白Wnt5a、CaMKII及NFAT的表达。综上,试验成功建立BAECs分离培养方法,BCG感染增加BAECs内Wnt5a表达和细胞自噬,抑制Wnt5a下调BCG诱导的BAECs细胞自噬,且Wnt5a是通过非经典Wnt/Ca²⁺信号通路调控BCG诱导的BAECs细胞自噬。     

IL-17细胞因子家族调节黏膜免疫的研究进展

皮肤和黏膜上皮细胞既是机体的物理屏障,又是机体防御微生物的第一道防线。上皮细胞与白细胞、树突细胞(DCs)等之间的相互作用,是机体产生适应性免疫反应的重要因素。IL-17细胞因子家族是最近新发现的具有强大的促炎症作用的细胞因子,它们在机体的固有和适应性免疫中发挥着重要作用,IL-17A、IL-17C和IL-17F能够直接作用于组织的上皮细胞,诱导各种免疫反应来对抗病原体,且能够促进组织的修复。IL-17E最基本的作用是作用于白细胞和诱导Ⅱ型免疫,这在其对抗寄生虫的作用中是非常关键的,此外,IL-17E还可以反向调节白细胞对IL-17A和IL-17F的产生;而对于IL-17B和IL-17D的研究则相对较少一些。     

IWF诱导小麦悬浮细胞H2O2迸发及其产生机制初探#br#

【目的】采用具有激发子活性的感染叶锈菌的小麦叶片细胞间隙液IWF-260刺激小麦洛夫林10悬浮细胞,探讨细胞感受激发子刺激引发的H2O2迸发情况及其产生的可能分子机制。【方法】采用化学发光法以及荧光探针DCFHDA、HE来研究H2O2的迸发以及NADPH氧化酶的活化。同时借助药物学试验探讨Ca2+和活性氧的关系。【结果】IWF-260可以引起悬浮细胞H2O2爆发,其在IWF-260刺激诱发的细胞死亡过程中发挥重要作用。而质膜NADPH氧化酶的激活以及胞外钙离子的内流与H2O2爆发有密切关系。【结论】IWF-260能够诱导细胞产生活性氧,质膜NADPH氧化酶和胞外钙离子内流参与了活性氧爆发过程。     

应用反相高效液相色谱法测定绵羊瘤胃液、肉鸡粪中吲哚和粪臭素含量

[目的]建立适于绵羊瘤胃液和肉鸡粪中痕量吲哚、粪臭素测定的反相高效液相色谱法.[方法]肉鸡日粮中分别添加饲用抗生素、葡萄糖酸和乳果糖,收集13～15、20～22、33～35和40 ～41日龄肉鸡的粪;采集绵羊饲喂前(记为0h),饲喂后1.5、3、6和9h的瘤胃内容物.[结果]色谱条件,流动相:乙腈:水,流速1mL/min,柱温30℃,激发波长270nm,发射波长350nm,梯度洗脱;方法具有分离时间短(10 min以内),样品处理简单、分离度好、灵敏度高(吲哚142.8 ng/mL,绝对量2.142 ng/15 μL;粪臭素1 ng/mL,绝对量0.015ng/15 μL)的特点.葡萄糖酸组、乳果糖组肉鸡粪中吲哚含量在肉鸡34～36日龄时达到高峰值,抗生素组肉鸡粪中吲哚含量随日龄呈现上升趋势;从粪臭素含量来看,40 ～41日龄时肉鸡粪中含量有所增加,其中,饲用抗生素组、乳果糖组增加最明显,葡萄糖酸组增加最少.绵羊瘤胃液中吲哚含量在饲喂后1.5h达到最高值,饲喂800 g精料日粮时含量极显著高于饲喂400 g精料日粮,其余各时间点两种日粮之间差异不显著;而瘤胃液中粪臭素含量在饲喂后3h达到最高值,且饲喂800 g精料日粮时含量都有高于饲喂400 g精料日粮时.[结论]肉鸡粪中的吲哚、粪臭素含量不仅与日粮有关,还与肉鸡的日龄有关.饲喂两种水平的精料补充料时绵羊瘤胃液中吲哚、粪臭素含量的动态变化规律基本相似;高精料饲喂后1.5 ～9 h内,瘤胃液中的粪臭素含量普遍升高.     

羊乳腺上皮细胞的形态观察

用组织块培养法获得山羊乳腺细胞原代培养物,并根据山羊乳腺成纤维细胞与上皮细胞对胰蛋白酶的敏感性不同将二者分离纯化。对细胞形态进行光镜观察发现:纯化的山羊乳腺上皮细胞传代至第15代时其生长仍正常。山羊乳腺上皮细胞含不同的细胞类型,大多数上皮细胞呈短梭形或多角形,呈蜂窝状;部分细胞呈圆饼状,体积较大;部分细胞呈长形。纯化的乳腺上皮细胞增殖可形成圆顶型结构,呈乳头状,称之为乳球体。     

Nodular dermatofibrosis and cystic renal disease in three mixed-breed dogs and a Boxer dog

Three mixed-breed dogs and a Boxer dog with nodular dermatofibrosis are described. The three mixed breed dogs had concurrent renal epithelial cysts ( n  = 2) or renal cystadenomas ( n  = 1); these lesions were visualized antemortem on ultrasonographic examination. One of these dogs was a 7-year-old Labrador Retriever-Gordon Setter mixed breed which survived 5 years after diagnosis, and was euthanized for unrelated causes. The Boxer dog had renal cystic adenomatous hyperplasia and renal cystadenocarcinomas which were not visualized on ultrasonographic examination but were diagnosed at necropsy.