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。     

猪繁殖与呼吸综合征病毒感染猪肺脏组织环腺苷酸磷酸二酯酶的活性及其mRNA表达变化

试验旨在探究在猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)感染过程中环腺苷酸(cAMP)特异性磷酸二酯酶(PDE)的活性及其mRNA表达量的变化,以期为PDE抑制剂的应用提供相关依据。试验采集PRRSV感染猪肺脏组织,运用高效液相色谱(HPLC)法检测cAMP在PDE反应前后的变化,计算cAMP-PDE活性;通过Real-time PCR检测cAMP-PDE mRNA表达量的变化。结果显示,PRRSV感染猪肺脏组织中cAMP-PDE活性显著高于对照组(P<0.05),在检测的8个PDE亚型中,除PDE4A外,PDE4B、PDE4C、PDE4D、PDE7A、PDE7B、PDE8A、PDE8B mRNA的表达量均高于对照组。结果表明,cAMP-PDE在PRRSV感染引起的猪肺脏部的炎症反应过程中存在异常变化,提示cAMP-PDE特异性抑制剂有望减轻PRRSV感染引起的猪肺脏部炎症损伤。     

中药"催情散"中硒含量分析

运用2,3-二氨基萘法通过环乙烷萃取(荧光法)测定了中药"催情散"煎剂以及其单药中硒含量.结果表明"催情散"中硒平均含量为0.071±0.0002mg/ml,处方中各单味药硒含量均超过一般植物体内全硒含量(0.1mg/kg),其中当归中硒含量最低(0.339±0.0032mg/kg),益母草中硒含量最高(1.370±0.028mg/kg)."催情散"处方煎剂及各单味药的高硒含量,为该处方的临床作用机制提供了新的解释途径.     

东北林蛙消化系统LDH同工酶表型的季节变化

为了解小兴安岭地区不同季节东北林蛙消化系统中食道、胃、肠道和肝脏的乳酸脱氢酶(LDH)同工酶的分布情况和活性,试验采用聚丙烯酰胺凝胶垂直板电泳对其进行分离分析。结果表明:东北林蛙消化系统4种器官组织中LDH同工酶共分离出电泳迁移率为0.299~0.153,基因型为C’4、CC’3、C2C’2、C3C’、C4、AB2C、B4、B’4、A2C2、A2B2、A2B’2、A3B、A4的LDH1~LDH13的13条谱带。4种器官组织在不同季节分离出的谱带数不同,在春季食道、胃、肠道和肝脏中均分离出11条谱带,夏季分别分离出10,10,11,9条谱带,秋季分别分离出4,4,5,5条谱带,冬季分别分离出7,6,6,6条谱带。电泳迁移率为0.255,0.242的LDH6和LDH7为冬季特有谱带。各组织中LDH13活性均最强。LDH同工酶的分布和活性具有明显的组织特异性,在不同季节的各组织器官中的表达程度不同,呈现多态性,具有季节性的差异。     

附子理中汤加减对母猪生产性能及血液理化指标的影响

试验旨在研究附子理中汤加减复方中药制剂对能繁母猪生产性能及血液理化指标的影响。选取96头有繁殖障碍史的“长×大”二元杂母猪，随机分为4组，每组4个重复，每个重复6头猪。试验A组、B组、C组分别在基础日粮中添加3%、2%、1%的附子理中汤加减复方中药制剂，D组（对照组）在基础日粮中添加0.5%抗生素。试验期150 d。结果显示，与对照组相比，B组母猪的平均产仔数显著升高（P<0.05）;3个中药组平均活仔数显著升高（P<0.05），仔猪出生均重、仔猪平均日增重极显著升高（P<0.01），平均木乃伊胎数、仔猪腹泻率均显著降低（P<0.05）。用药前，除单核细胞计数（MONO）、中性粒细胞（GRAN）外，其他各项血液指标均异常。用药后，各组猪的血红蛋白浓度（HGB）、血细胞比容（HCT）均恢复正常值；3个中药组猪的白细胞计数（WBC）均恢复正常值，对照组母猪仍处于异常；A组和B组猪的红细胞计数（RBC）均恢复正常值，C组和对照组处于异常；A组猪平均红细胞血红蛋白含量（MCHC）恢复正常值，对照组仍处于异常。用药前，除血清白蛋白（ALB）、白球比（A/G）、尿素（URE...     

孕鼠暴露双酚A对仔鼠存活率、生殖激素及相关基因的影响

为了研究孕鼠在孕期暴露双酚A（bisphenol A,BPA）对仔鼠生殖激素及相关基因的影响,试验将40只昆明孕鼠随机分为A、B、C、D共4组,每组10只。其中A组为对照组,饲喂普通鼠粮;B、C、D组孕鼠整个妊娠期（妊娠1 d至分娩）分别按每只鼠每天50、500、2 500 mg/kg体重给予BPA,待孕鼠自然分娩,观察记录仔鼠死亡情况。至仔鼠性成熟（56日龄）剖杀仔鼠,摘取睾丸或卵巢称重并计算脏器指数,HE染色观察卵巢或睾丸组织结构的变化,ELISA试剂盒分析仔鼠血清睾酮（T）、促卵泡素（FSH）及雌二醇（E₂）水平,免疫组织化学方法检测仔鼠睾丸或卵巢Bax、Bcl-2蛋白的表达,实时荧光定量PCR检测仔鼠睾丸StAR、CYP11a或卵巢AMH、Kitlg mRNA表达。结果显示,孕鼠暴露BPA极显著增加了仔鼠死亡率（P<0.01）,显著降低了仔鼠睾丸重（P<0.05）。ELISA检测结果表明,孕鼠暴露BPA极显著降低了仔鼠T（♂）及FSH（♀）含量（P<0.01）,极显著升高了仔鼠（♀） E₂水平（P<0.01）。HE染色结果显示,随BPA剂量增加,仔鼠睾丸组织损伤严重,间质细胞减少;卵巢组织结构随BPA剂量增大,空泡逐渐增多,黄体颗粒数量逐渐减少。免疫组化结果显示,孕鼠暴露BPA增加了仔鼠睾丸或卵巢组织中Bax阳性蛋白表达,减少了Bcl-2阳性蛋白表达,显著降低了雄性仔鼠StAR mRNA表达量（P<0.05）;B、D组雄性仔鼠CYP11a mRNA表达量极显著降低（P<0.01）,而C组CYP11a mRNA表达量极显著升高（P<0.01）;C、D组雌鼠Kitlg mRNA表达极显著降低（P<0.01）,AMH mRNA表达量显著升高（P<0.05）。本试验结果表明,孕鼠妊娠期暴露不同剂量BPA增加了仔鼠死亡率,扰乱了生殖激素平衡和睾丸/卵巢中相关凋亡蛋白及生殖基因表达。     

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。     

不同中药方剂对弓形虫感染小鼠脾细胞分泌 IL-2活性的影响

通过观察不同方剂对小鼠感染弓形虫后脾 IL-2含量的影响,确定对弓形虫病治疗效果最佳的复方中药。将昆明系小鼠随机分为中药复方方剂组（A、B、C 组）、复方 SMZ 组（D 组）、模型组（E 组）和对照组（F 组）。用 RH 株弓形虫速殖子腹腔感染小鼠,2 h 后灌胃给药,其中 A、B、C 3个组每只每次0．5 mL 自拟弓形虫汤剂;D 组每只每次0．5 mL 复方 SMZ 混悬液300 mg／（kg· d－1）,疗程15 d。采用双抗夹心ELISA 法,观察不同方剂对小鼠脾细胞 IL-2含量的影响,并对照不同方剂之间的差异,采用 F 检验和 q 检验进行统计分析。结果表明,A、B、C 组和 D 组 IL-2活性水平均随着时间延长而逐渐升高;A、C、D 组与 F组有显著性差异;B 组 IL-2水平活性保持了较高值,与 E 组差异极显著。说明3种自拟中药复方方剂均能显著提高弓形虫感染小鼠脾细胞 IL-2的含量,增强小鼠免疫功能,其中复方二提高最显著,对 IL-2活性影响最大。     

鸡褪黑激素受体基因在开产前后不同组织中的表达差异

为了从分子水平上了解鸡褪黑激素受体（MTNR）基因在开产前后不同组织中的表达差异,以β-actin基因为内标基因,运用实时荧光定量PCR方法,比较了MTNR1A、MTNR1B和MTNR1C这3个受体基因在心脏、肝脏、输卵管、卵巢、脑和视交叉各组织及在鸡开产前后表达量的差异。结果表明,MTNR1A只在开产后各组织中的表达量存在差异,其中以卵巢组织表达量最高,心脏中表达量最低。MTNR1B和MTNR1C的表达量在开产前或开产后均表现出组织间的显著差异;在开产前后的各组织中,MTNR1B的表达量均以视交叉为最高,卵巢为最低;而MTNR1C的表达量在开产前以心脏中最高,开产后在视交叉中最高,在卵巢组织中的表达量开产前后均为最低。同一组织开产后与开产前比较,MTNR1C基因在心脏、输卵管和脑组织中的表达量呈显著下调趋势。因此,3个受体基因在鸡的不同组织和生理时期存在表达差异。     

大量使用非常规饲料对育肥猪生产性能的影响

试验组A饲料中添加非常规饲料、试验组B在饲料中添加中药添加剂。试验组C在饲料中添加苜蓿草。测定试验各组胴体品质、猪肉风味和品质等各项指标。试验结果表明：平均末重、日增重上差异极显著（P〈0．01）。A、B、C3组在中猪期的料肉比和在大猪期的料肉比以及全期的料肉比差异均极显著（P〈0．01）。     

5种青藏高原中药材对磷酸二脂酶4活性的影响

目的：从青藏高原地产中药材中筛选磷酸二脂酶4（PDE4）特异性抑制剂。方法：选取甘草、羌活、柴胡、大黄、红景天5种药材为试验对象,以PDE4特异性抑制剂Rolipram为阳性对照组,从猪中性粒细胞中提取PDE4进行PDE4活性实验。结果：5 mg/mL甘草、羌活、柴胡、大黄、红景天水提物、10μmol/L Rolipram组PDE4活性分别为6.8%、11.02%、11.07%、12.7%、18.7%、7.61%;抑制率分别为38.18%、0.02%、-0.47%、-15.21%、-69.66%、30.90%。与空白对照组PDE4活性相比较,甘草组P〈0.05;红景天组P〈0.001。与Rolipram组抑制率相比较,柴胡组P〈0.05;大黄组P〈0.01;红景天组P〈0.001。甘草组对PDE4抑制率高于PDE4特异性抑制剂Rolipram。结论：5mg/mL甘草水提物能显著抑制PDE4活性,且高于PDE4特异性抑制剂Rolipram,5 mg/mL柴胡、羌活水提物对PDE4活性的影响不明显,5 mg/mL大黄水提物对PDE4的活性具有促进作用,5 mg/mL红景天对PDE4的活性具有极显著的促进作用。     

猪JHDM2A基因的克隆及表达分析

试验旨在克隆猪JHDM2A基因,并研究其在猪卵巢组织中的表达情况。首先克隆猪JHDM2A基因,并构建pLVX-IRES-ZsGreen1-JHDM2A真核表达载体,同时对JHDM2A基因在猪卵泡发育过程中的表达情况进行分析。结果显示,克隆得到的猪JHDM2A基因编码区长度为3 945 bp,编码1 315个氨基酸。通过多重氨基酸序列比对发现,猪JHDM2A基因与黄牛、水牛、绵羊和人相应氨基酸序列的同源性分别为93.5%、94.7%、94.7%和93.8%。蛋白质分子系统进化树分析结果表明,JHDM2A基因在物种进化过程中高度保守。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-JHDM2A真核表达载体导入HEK293T细胞,可观察到清晰的绿色荧光蛋白表达。免疫组化结果显示,JHDM2A蛋白在不同发育阶段猪卵泡中均有表达。本试验通过克隆获得猪JHDM2A基因序列,JHDM2A蛋白在猪卵巢中高度表达,表明其功能可能与猪卵泡发育密切相关。     

Cloning and Expression Analysis of Porcine JHDM2A Gene

The aim of this study was to clone the JHDM2A gene of porcine and study the expression of JHDM2A gene in porcine ovary. In this study, we cloned the porcine JHDM2A gene and constructed its eukaryotic expression vector, the expression of JHDM2A gene in porcine ovarian tissue was also analyzed. The results showed that the cloned CDS length of porcine JHDM2A gene was 3 945 bp, which encoded 1 315 amino acids. The results of multiple amino acid sequence comparison showed that the porcine JHDM2A shared 93.5%, 94.7%, 94.7% and 93.8% homologous compared with those of Bubalus bubalis, Bos taurus, Ovis aries and Homo sapiens, respectively. Phylogenetic tree analysis indicated that the JHDM2A gene was highly conservative in the evolutionary process. The pLVX-IRES-ZsGreen1-JHDM2A eukaryotic expression vector was constructed, and clear green fluorescent signal was observed when the plasmid was transfected into HEK293T cells by liposome method. The immunohistochemical results showed that the JHDM2A protein was expressed in porcine follicle of different development stages.The results showed that the porcine JHDM2A gene sequences was cloned, and the JHDM2A protein was highly expressed in porcine ovary, indicating that its function might be closely related to the development of porcine follicular.     

Phosphodiesterase isoenzymes in equine platelets and their influence on platelet adhesion

OBJECTIVE: To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion. SAMPLE POPULATION: Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean +/- SEM, 17.3 +/- 1.1 years). PROCEDURES: PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay. RESULTS: PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PDE3 abolished platelet adhesion almost completely, whereas inhibition of PDE4 and PDE5 had little effect. CONCLUSIONS AND CLINICAL RELEVANCE: Function of equine platelets can be influenced by inhibition of PDE3. Selective PDE3 inhibitors may be clinically useful to regulate platelet function. They offer the advantage of increased potency with fewer adverse effects, compared with those for nonselective PDE inhibitors.     

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic‐polyadenylation‐element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT‐PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant‐negative mutant pCPEB1 (AA‐CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA‐CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.     

In vitro and In vivo Association of Porcine Hepatic Cytochrome P450 3A and 2C Activities with Testicular Steroids

The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β‐estradiol, 17α‐estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism‐based inhibitions were examined. 7‐benzyloxyresorufin (BR) and 7‐benzyloxy‐4‐trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7‐benzyloxyresorufin O‐dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism‐based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β‐oestradiol. Mechanism‐based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α‐oestradiol through the mechanism‐based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.