鱼类促性腺激素抑制激素及其受体的研究进展

促性腺激素抑制激素是2000年由日本学者首次从鹌鹑脑中分离出的一种新型下丘脑神经肽,通过其受体介导参与机体的生长、生殖以及摄食等生理过程。迄今,只在金鱼、斑马鱼、星点东方鲀、罗非鱼以及斜带石斑鱼等几种鱼中鉴定出促性腺激素抑制激素。目前,鱼类促性腺激素抑制激素的生理学功能研究相对较少,且存在争议。鱼类促性腺激素抑制激素及其受体的表达调控以及其他生理学功能仍有待进一步研究。本研究简要总结鱼类促性腺激素抑制激素及其受体的研究进展,并对促性腺激素抑制激素的生理学功能进行概括讨论,旨在加深对鱼类促性腺激素抑制激素的认识和了解,为进一步研究做铺垫。     

团头鲂促肾上腺皮质激素释放激素受体基因序列特征及表达分析

促肾上腺皮质激素释放激素受体(corticotropin-releasing hormone receptor,CRHR)是鱼类下丘脑–垂体–头肾调控轴上的重要应激调节因子。本研究通过c DNA末端快速扩增(RACE)技术成功克隆出团头鲂(Megalobrama amblycephala)CRHR1 m RNA全长序列,应用生物信息学方法对其序列特征进行解析;同时采用荧光定量PCR技术分析了团头鲂CRHR1的组织分布图谱及外源性皮质醇注射模拟应激处理下团头鲂CRHR1 m RNA的表达变化。研究结果表明,CRHR1 m RNA序列开放阅读框(open reading frame,ORF)为1290 bp,编码429个氨基酸。团头鲂CRHR1氨基酸序列与鲤科鱼类的CRHR1氨基酸序列同源性最高;该受体具有7次横跨膜结构和氨基端激素受体结构域。CRHR1在垂体中表达量最高,其次为下丘脑,在心脏、肝、脾等组织中表达丰度较低。经外源性皮质醇注射后,实验组血糖和血清皮质醇显著高于对照组,在处理2 h后达到峰值;实验组血清ACTH水平与对照组差异总体不显著,但呈现先升高后下降。应激处理后,CRHR1转录水平在4种组织中的表达变化存在差异;垂体中CRHR1在早期出现明显的表达抑制,在下丘脑中则呈现先缓慢升高后缓慢下降的趋势,而心脏和头肾中CRHR1在早期则表现出表达迅速上调而后缓慢下降的趋势。本研究进一步丰富了CRHR在鱼类研究方面的基础资料,为鱼类应激调控提供了理论基础。     

水杨酰苯胺类mglu5拮抗剂的设计、合成及活性研究

目的 利用骨架跃迁思想设计并合成新型水杨酰苯胺类代谢型谷氨酸受体5（metabotropic glutamate receptor 5, mglu5）拮抗剂。方法 将取代水杨酸与二取代苯胺缩合得目标化合物I-IV,用核磁共振和质谱进行结构确证,荧光测钙流法进行拮抗mglu5活性测定。结果 设计的目标化合物对mglu5具有明显的拮抗活性,其中化合物I拮抗活性最好,IC₅₀为0.079 μmol/L。结论 水杨酰苯胺类化合物为潜在的新型mglu5拮抗剂,值得进一步研究。     

百脉根LysM型受体LcLRK1参与菌根真菌丛枝形成

从日本百脉根(Lotus corniculatus)中克隆到一个可能与菌根因子识别相关的Lc LRK1基因,通过设计简并引物并利用RACE PCR手段,在百脉根中扩增得到Lc LRK1基因全长序列。通过生物信息学的方法,对Lc LRK1基因进行结构分析和预测;接种Rhizophagus irregularis,检测Lc LRK1基因在百脉根不同组织中的表达水平;构建Pro Lc LRK1∶∶GUS重组载体,观察Lc LRK1基因在根中的表达部位;最后通过RNA干扰(RNAi)技术进行Lc LRK1基因沉默。结果表明:Lc LRK1编码一个Lys M型受体激酶,由3个胞外Lys M结构、1个跨膜结构域与1个胞内激酶结构域组成;Lc LRK1在AM共生早期表达水平明显增强,并在根的表皮细胞及根毛基部表达;Lc LRK1基因沉默后抑制了早期丛枝的形成;相对于对照组,在Lc LRK1RNAi的植株中AM真菌的侵染率明显下降,丛枝数目也明显降低。证实Lc LRK1基因与菌根真菌信号分子识别相关,并且影响AM真菌丛枝的形成。     

Steroids with inhibitory activity against the prostate cancer cells and chemical diversity of marine alga Tydemania expeditionis

A new diketosteroid, (E)-stigmasta-24(28)-en-3,6-dione (1), along with three known steroids (2-4) was isolated from marine alga Tydemania expeditionis collected in China Sea. Their structures were elucidated by extensive spectroscopic methods. Comparison of the chemical constituents revealed significant diversity among different locations. The biological activities of 1, 3 and 4 were evaluated on the prostate cancer cell lines and androgen receptor. Compound 1 exhibited moderate inhibitory activities against the prostate cancer cells DU145, PC3 and LNCaP with IC(50) values of 31.27±1.50, 40.59±3.10 and 19.80±3.84 μM, respectively. Compound 3 showed more potent activities with IC(50) values of 12.38±2.47, 2.14±0.33 and 1.38±0.07 μM, respectively. However, compound 4 showed only weak inhibitory activities on LNCaP cells and was inactive on DU145 and PC3 cells. A competitive binding assay showed that compound 1 exhibited significant affinity to the androgen receptor with an IC(50) value of 7.19±0.45 μM, while 3 and 4 were inactive. The fact that the inhibitory properties of 1 and 3 against the prostate cancer cells were inconsistent with their affinities to the androgen receptor suggested that there might be other mechanism of action involved in the cytotoxic activity.     

FcγRⅡB介导的PRRSV感染的抗体依赖性增强作用

将PRRSV Hn-1/06株阳性血清（中和效价为1∶2）做2～8倍倍比稀释后,分别与等体积含100TCID50/100μL的PRRSV Hn-1/06株病毒液混合,制备感染性免疫复合物。取纯化的猪IgG和兔抗猪IgG制备非感染性免疫复合物。用鼠抗FcγRⅡB阳性血清封闭猪肺泡巨噬细胞（PAM细胞）表面的FcγRⅡB,然后分别将感染性免疫复合物和非感染性免疫复合物与PRRSV的混合物感染封闭后的PAM细胞,培养48h后收获样品,采用荧光定量RT-PCR检测收获样品中PRRSV的含量。结果表明,选择性封闭PAM细胞的FcγRⅡB能增强PRRSV的抗体依赖性增强作用。     

冬枣果实中1个ETR2类乙烯受体基因的克隆及其表达

冬枣(Ziziphus jujuba‘Dongzao’)是我国的特色鲜食果品之一,果实富含维生素C和环磷酸腺苷等成分,具有较高的营养和药用价值。冬枣采后贮藏过程中极易失水、皱缩、酒软或霉烂等,致使其供应期甚短。乙烯的生理作用是由位于细胞内质网上乙烯受     

小尾寒羊、无角道赛特及其杂交后代BMPR-IB基因多态性研究

为了检测骨骼形态发生蛋白IB型受体(BMPR-IB)基因在小尾寒羊母羊、无角道赛特公羊、道×寒F1和F2代杂交公羊及道×寒F2横交固定所产种公羊中的多态性,分析小尾寒羊基因型与产羔数的相关性,试验采用PCR-RFLP法对BMPR-IB基因进行分型。结果表明:在无角道赛特公羊中只有1种基因型,即野生型(++);在小尾寒羊母羊中检测到2种基因型(BB和B+),基因型频率分别为0.35,0.65;在道×寒F1和F2代杂交公羊中检测到2种基因型(B+和++),F1代基因型频率分别为0.75,0.25,F2代基因型频率分别为0.30,0.70。小尾寒羊的2种基因型产羔数差异极显著(P<0.01),BMPR-IB基因的遗传方式符合孟德尔遗传定律。     

兔TLR2、TLR3和TLR4部分cDNA序列的克隆及分析

用RT-PCR技术从日本大耳白兔脾脏组织克隆出兔Toll样受体2、3、4基因（拟命名为RTLR2、R TLR3和RTLR4）的cDNA序列并进行测序,获得的3个Toll样受体基因序列长分别为128、150和139bp,并将其测序结果与GenBank中登录的穴兔（Oryctolagus cuniculus）的Toils核苷酸序列进行比对,发现本次克隆到的RTLR2与穴兔TLR2的基因序列（NM_001082781）相似性为99％,而RTLR3与TLR3（NM_001082219）和RTLR4与TLR4（NM_001082732）相似性均为100％。Protein Blast同源性结果显示,RTLR2、RTLR3和RTLR4的氨基酸序列与穴兔TLR2、TLR3和TLR4的同源性皆为100％,与马、人、野猪等其他8种动物TLRs的比较,与RTLR2同源性最高的是马的80％,其他的只有61％～78％;与RTLR3同源性最高是马和野猪的97％,其他也较高达93％～95％;而RTLR4与其他8种动物的同源性均较低75％以下。结果表明：RTLR2、RTLR3和RTLR4分别为免TLR2、TLR3和TLR4的部分cDNA基因序列;TLRs在不同物种的进化过程中存在种属特异性。     

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar

In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg(-1) d(-1) was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen-estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.