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。     

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。     

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。     

五指山猪与长白猪65 d胎儿组织MRFs家族基因的表达研究

 
以五指山猪和长白猪妊娠65 d胎儿腿部骨骼肌、背部骨骼肌、心脏、肝脏、脾脏、肺脏、肾脏、肠、胃、脑10种组织为材料,采用qPCR技术,检测和研究MRFs基因家族成员（包括MyoD1、Myf4、Myf5和Mfy6基因）在以上10种组织中的mRNA表达水平及差异。结果表明：（1）MRFs家族成员除肝、肾、肠组织以外,在五指山猪65 d猪胎儿组织中mRNA表达水平均高于长白猪相应组织中的表达水平（P<0.05）;（2）MRFs家族成员表达水平在腿肌和背肌中的表达普遍偏高,其他组织中表达较低（P<0.05）;（3）2个猪种在以上10种组织中MRFs家族mRNA表达水平的高低分布基本一致,其相对表达量顺序为：腿部骨骼肌＞背部骨骼肌＞肝＞肺＞胃＞脾＞脑＞肾＞肠＞心。     

STC-1基因在大、小体型猪中表达与分布的差异分析

试验分别采集40日龄小体型猪（巴马猪）和大体型猪（大白猪）的心脏、肝脏、脾脏、肺脏、肾脏、头骨、骨骼肌组织,利用实时荧光定量PCR检测斯钙素-1（stanniocalcin 1,STC-1）基因mRNA在各个组织中的表达水平,并通过Western blotting检测STC-1蛋白在各个组织中的分布。实时荧光定量PCR检测结果表明,STC-1基因mRNA在巴马猪和大白猪肺脏、肾脏中相对表达水平较高,在骨骼肌中的表达水平最低;除心脏和骨骼肌外,巴马猪其余各组织中STC-1基因mRNA表达水平均显著高于大白猪（P < 0.05）。Western blotting检测结果表明,巴马猪肝脏中STC-1蛋白的表达量最高,而大白猪脾脏中STC-1蛋白表达量最高,两者差异显著（P < 0.05）;巴马猪肺脏、肝脏、骨骼肌及心脏组织中STC-1蛋白表达量均极显著高于大白猪（P < 0.01）;而巴马猪肾脏、脾脏中STC-1蛋白表达量极显著低于大白猪（P < 0.01）。本研究首次对大、小体型猪不同组织的STC-1基因mRNA表达水平及其STC-1蛋白分布进行检测,导致该基因表达与分布差异的原因可能与两种猪受外界环境应激及生长发育差异有关。     

m6A甲基化在鸡肌肉生长发育中的表达研究

试验旨在研究RNA m6A修饰相关基因去甲基化酶Alk B同源蛋白5（Alk B homologue 5，ALKBH5）、去甲基化酶肥胖相关蛋白（fat mass and obesity-associated protein，FTO）、甲基转移酶样蛋白3（methyltransferase like 3，METTL3）、甲基转移酶样蛋白14（methyltransferase like 14，METTL14）和成肾细胞瘤1-结合蛋白（Wilms’tumor 1-associating protein，WTAP）在鸡骨骼肌发育过程中的表达，分析其与骨骼肌m6A甲基化水平的相关性。首先，利用实时荧光定量PCR技术检测m6A甲基化相关基因在金茅花鸡12（E12）、14（E14）、16（E16）、18（E18）胚龄和1日龄腿肌和胸肌组织中mRNA表达水平，以及其在鸡成肌细胞50%、100%增殖期和1、2、3、4、5 d分化期的mRNA表达水平；随后，利用m6A甲基化试剂盒检测金茅花鸡E12和1日龄腿肌和胸肌组织中m6A甲基化修饰水平，与m6A甲基化相关基因表达水平进行相关性分析。结果显示，m6A去甲基化基因ALKBH5和FTO mRNA表达水平在骨骼肌发育过程中显著上调（P<0.05），即在E12、E14低表达，E16、E18逐渐上调，1日龄达到最高。m6A甲基化写入基因METTL14、METTL3和WTAP mRNA表达水平在E12、E14、E16逐渐上升，E18下降，随后至1日龄表达量回升。在细胞增殖过程中，ALKBH5、FTO、METTL14、METTL3和WTAP基因表达均上调；在细胞分化过程中ALKBH5和FTO基因表达水平显著上调（P<0.05），在分化第5天达到最高。METTL14、METTL3和WTAP基因mRNA表达水平在细胞诱导分化的1、2、3、4 d表达量呈下降趋势，而在诱导分化的第5天有所回升。甲基化水平检测结果显示，腿肌和胸肌m6A甲基化水平变化趋势一致，均在胚胎发育过程中显著下降（P<0.05），至1日龄达到最低。相关性分析结果显示，鸡骨骼肌RNA m6A甲基化水平与m6A去甲基化修饰基因ALKBH5、FTO mRNA表达水平呈显著负相关（P<0.05）。综合以上试验结果，推测m6A甲基化修饰与鸡骨骼肌发育相关，而去甲基化基因ALKBH5、FTO可能通过调控RNA m6A甲基化水平，影响鸡骨骼肌发育。本研究结果为进一步研究m6A甲基化修饰调控鸡骨骼肌生长发育的功能和分子机制提供理论依据。     

CYP7B1和CYP4B1基因mRNA在不同毛色贵州剑白香猪皮肤组织中的表达分析

为了分析CYP7B1、CYP4B1基因在剑白香猪的黑色和白色皮肤组织中的表达差异,试验通过提取剑白香猪黑色和白色皮肤组织的RNA,分别逆转录合成相应的cDNA;以GAPDH基因作为内参,采用实时荧光定量PCR (qRT-PCR)检测CYP7B1、CYP4B1基因mRNA在黑色和白色皮肤组织中的相对表达量。结果:CYP7B1、CYP4B1基因在剑白香猪黑色皮肤中表达量较高,在白色皮肤组织中表达量很少甚至几乎不表达。结论:推测CYP7B1、CYP4B1基因可能与剑白香猪黑色皮肤的形成有关,为进一步研究CYP7B1、CYP4B1基因在香猪的黑色和白色皮肤组织中的功能提供数据基础。     

过表达KDM4家族基因对猪克隆胚胎体外发育效率的影响

为了探究过表达H3K9me3的组蛋白去甲基化酶4(KDM4)家族基因对猪克隆胚胎体外发育效率的影响，试验采用pc-KDM4A～D质粒构建、酶切鉴定、细胞转染、定量PCR扩增及体外转录等方法制备过表达KDM4A～D的猪胎儿成纤维细胞和KDM4A～D mRNAs,通过体细胞核移植和显微注射的方式制备过表达KDM4A～D的猪克隆胚胎。将供体细胞过表达KDM4A～D的猪克隆胚胎分为1个对照组和5个试验组，分别为pcDNA3.1(+)组、pc-KDM4A组、pc-KDM4B组、pc-KDM4C组、pc-KDM4D组及4质粒组；将注射KDM4A～D mRNA的猪克隆胚胎分为1个对照组和5个试验组，分别为H₂O组、KDM4A组、KDM4B组、KDM4C组、KDM4D组及4mRNA组。统计各组的卵裂数、囊胚数和囊胚细胞数；利用免疫荧光检测4质粒组和4mRNA组4-细胞期猪克隆胚胎中H3K9me3的表达水平。结果表明：pc-KDM4A～D质粒构建成功，质粒转染的猪胎儿成纤维细胞可过表达KDM4A～D和降低细胞内H3K9me3水平。与pcDNA3.1(+)组比较，4质粒组可显著提高猪...     

不同品种猪在不同生理阶段骨骼肌葡萄糖转运载体和胰岛素受体表达的发育性变化

本试验旨在研究不同品种猪在不同生理阶段骨骼肌葡萄糖转运载体(GLUT)1、GLUT4和胰岛素受体(IR)mRNA表达的发育规律。试验以1、7、30、60、90和150日龄蓝塘和长白两品种猪背最长肌、半膜肌和半腱肌组织样品c DNA为模板,采用实时荧光半定量PCR的方法,检测猪GLUT1和GLUT4 mRNA在不同骨骼肌中的表达模式。结果显示:1)1~150日龄蓝塘和长白猪背最长肌、半膜肌和半腱肌GLUT1 mRNA表达存在相似的发育变化规律,在1日龄时相对表达丰度最高。2)1~150日龄蓝塘和长白猪半膜肌和半腱肌GLUT4 mRNA相对表达丰度随日龄呈波形式变化,变化速度和程度存在品种差异。1日龄和150日龄时蓝塘猪背最长肌GLUT4mRNA相对表达丰度低于同日龄长白猪(P0.05),而在其余日龄时高于同日龄长白猪(P0.05)。3)蓝塘和长白猪背最长肌、半膜肌和半腱肌IR mRNA相对表达丰度在1和30日龄时表现出较高水平,而在7、60、90和150日龄时无显著差异(P0.05);同日龄蓝塘和长白猪之间IR mRNA相对表达丰度无显著性差异(P0.05)。结果表明:猪骨骼肌GLUT1 mRNA的表达在不同生理阶段存在差异,猪骨骼肌GLUT4 mRNA的表达在不同品种和生理阶段存在显著差异,且其与IR mRNA的表达相关性较弱,无明显规律。     

不同品种猪在不同生理阶段骨骼肌葡萄糖转运体和胰岛素受体表达的发育性变化

本试验旨在研究不同品种猪在不同生理阶段骨骼肌葡萄糖转运载体(GLUT)1、GLUT4和胰岛素受体(IR)mRNA表达的发育规律.试验以1、7、30、60、90和150日龄蓝塘和长白两品种猪背最长肌、半膜肌和半腱肌组织样品cDNA为模板,采用实时荧光半定量PCR的方法,检测猪GLUT1和GLUT4 mRNA在不同骨骼肌中的表达模式.结果显示:1)1～ 150日龄蓝塘和长白猪背最长肌、半膜肌和半腱肌GLUT1 mRNA表达存在相似的发育变化规律,在1日龄时相对表达丰度最高.2)1 ～ 150日龄蓝塘和长白猪半膜肌和半腱肌GLUT4 mRNA相对表达丰度随日龄呈波形式变化,变化速度和程度存在品种差异.1日龄和150日龄时蓝塘猪背最长肌GLUT4mRNA相对表达丰度低于同日龄长白猪(P>0.05),而在其余日龄时高于同日龄长白猪(P>0.05).3)蓝塘和长白猪背最长肌、半膜肌和半腱肌IR mRNA相对表达丰度在1和30日龄时表现出较高水平,而在7、60、90和150日龄时无显著差异(P>0.05);同日龄蓝塘和长白猪之间IR mRNA相对表达丰度无显著性差异(P>0.05).结果表明:猪骨骼肌GLUT1 mRNA的表达在不同生理阶段存在差异,猪骨骼肌GLUT4 mRNA的表达在不同品种和生理阶段存在显著差异,且其与IR mRNA的表达相关性较弱,无明显规律.     

Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.     

Characterisation of glucose transporter (GLUT) gene expression in broiler chickens

1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.     

五指山猪不同组织中Myf5与MyoD1基因的表达研究

 MRFs家族成员包括Myf5、MyoD1、Myf4和Mfy6,利用Real time PCR技术,检测Myf5、MyoD1基因在从出生到体成熟（30 d、210 d、360 d）五指山猪背部肌肉组织中的表达变化趋势,以及Myf5、MyoD1基因在体成熟五指山猪心脏、肝脏、肺脏、脾脏、肾脏、肌、胃和小肠以上组织中的mRNA表达水平。结果表明：Myf5和MyoD1基因在出生后五指山猪背部肌肉组织中的mRNA表达水平与五指山猪的生长年龄成正比（P<0.05）;Myf5及MyoD1基因在成年五指山猪以上8种组织中均有表达,其中在肌肉组织中相对表达量最高。     

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.     

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。     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Expression and Distribution Difference Analysis of STC-1 Gene in Big and Small Body Shape Pigs

The heart,liver,spleen,lung,kidney,skull,skeletal muscle were collected from 40-day-old Bama Mini pig and Large White pig,stanniocalcin 1(STC-1) gene mRNA and STC-1 protein were detected with Real-time PCR and Western blotting methods, respectively. The Real-time PCR results showed that the expression level of STC-1 gene mRNA was higher in lung and kidney, but was the lowest in skeletal muscle of Bama Mini pig and Large White pig. Except for heart and skeletal muscle tissues, the expression level of STC-1 gene mRNA in other tissues of Bama Mini pig was significantly higher than Large White pig (P < 0.05).The Western blotting results showed that the STC-1 protein distribution were the highest in liver and spleen of Bama Mini pig and Large White pig, respectively, and the difference was significant (P < 0.05). The STC-1 protein expression in lung, liver, skeletal muscle and heart of Bama Mini pig were extremely significantly higher than Large White pig (P < 0.01),and the STC-1 protein expression in kidney and spleen of Bama Mini pig were extremely significantly lower than Large White pig (P < 0.01). STC-1 gene mRNA and protein were firstly detected in different tissues from big and small body shape pigs,this might have a relationship with various external environments stress and development.