谷氨酸钠诱导不孕大鼠肥胖无排卵机制探讨

目的探讨谷氨酸钠诱导大鼠肥胖后对其不孕的影响。方法给新出生雌性Wistar大鼠皮下注射谷氨酸钠,连续5d,以建立肥胖模型。观察神经肽Y(NPY)、瘦素受体(obR)及促性腺激素释放激素(GnRH)在其下丘脑弓状核的表达的变化;并测定血清雌二醇(E2)、睾酮(T)、瘦素(leptin)、卵泡刺激素(FSH)、黄体生成素(LH)的变化。结果80﹪的注射谷氨酸钠大鼠呈能量失衡致肥胖状态;血清E2、T、leptin水平较对照组明显升高(P<0.05),FSH、LH水平较对照组明显降低(P<0.05);下丘脑弓状核NPY表达较对照组增强(P<0.05),而obR及GnRH表达较对照组减弱(P<0.05)。结论谷氨酸钠诱导肥胖大鼠由于神经内分泌-代谢失调而引起GnRH水平降低,导致下丘脑-垂体-卵巢轴功能失调性无排卵而引起不孕。     

Kisspeptin/GPR54信号通路促使性早熟形成的作用观察

目的:以达那唑诱导的雌性性早熟模型大鼠为研究对象,探讨下丘脑kisspeptin/GPR54信号通路在性早熟发病机制中的作用。方法:3日龄SD雌性大鼠,随机分为正常组、对照组和模型组。于大鼠5日龄时,模型组皮下注射溶有达那唑的乙醇与乙二醇的混合液进行造模,分别于大鼠15、25、30、35和40日龄时处死,留取标本。观察性器官发育情况;采用ELISA法检测外周血中E2、FSH和LH水平;采用real-time PCR法检测下丘脑Kiss-1、GPR54和GnRH的mRNA表达;应用免疫荧光法观察下丘脑kisspeptin的表达情况。结果:模型大鼠青春期启动和性成熟时间较正常组和对照组显著提前(P0.05);在25和30日龄时,模型大鼠外周血清性激素水平和子宫系数均显著高于正常组和对照组(P0.05);在25和30日龄时,模型大鼠卵巢形态学发育也明显早于正常组;在25日龄时,模型组大鼠下丘脑Kiss-1和GnRH的mRNA及下丘脑弓状核kisspeptin表达显著高于正常组和对照组(P0.05);在30日龄时,模型组大鼠下丘脑弓状核kisspeptin表达低于正常组和对照组(P0.05);在35日龄时,模型组大鼠Kiss-1和GnRH的mRNA表达较正常组和对照组降低(P0.05);在所观察日龄内,各组大鼠GPR54mRNA的表达均无显著差异。结论:性早熟模型大鼠在青春期启动时下丘脑Kiss-1 mRNA和kisspeptin的表达显著上调,提示kisspeptin可能是性早熟形成的启动因子,kisspeptin/GPR54信号通路在性早熟的发病中可能起着重要作用。     

营养性肥胖大鼠弓状核促性腺激素释放激素的表达变化以及对精子发生的影响

目的:探讨营养性肥胖大鼠弓状核神经肽Y(NPY)、瘦素受体(ob-R)及与生殖相关的促性腺激素释放激素(GnRH)表达变化以及对精子发生的影响.方法:免疫组织化学观察NPY、ob R及GnRH在肥胖模型组下丘脑弓状核的表达情况以及睾丸支持细胞雄激素结合蛋白(ABP)表达变化;流式细胞分析检测睾丸生精细胞周期的改变.并测定血清中瘦素、睾酮、卵泡刺激素(FSH)和黄体生成素(LH)的水平.结果:肥胖大鼠血清中瘦素水平较对照组明显升高,睾酮、FSH、LH水平较对照组明显降低;下丘脑弓状核NPY表达较对照组增强,ob-R及GnRH表达较对照组减弱,ABP表达较对照组减弱;肥胖大鼠S期细胞显著下降,G2/M期细胞的百分数明显增多.结论:营养性肥胖大鼠由于神经内分泌代谢失调而引起GnRH水平降低,导致下丘脑垂体睾丸轴功能失调引起睾丸间质细胞及支持细胞功能降低,致使精子发生障碍,可能导致不育.     

电针对单纯性肥胖大鼠无排卵机制的影响

目的:探讨电针对食源性肥胖大鼠排卵机制的影响.方法:将刚断乳雌性SD大鼠分为2组,正常组喂以普通饲料,高能饲料组给予高能饲料,筛选出肥胖不孕大鼠.检测血清雌二醇(E2)、睾酮(T)、瘦素(leptin)、卵泡刺激素(FSH)、黄体生成素(LH)的水平;观察神经肽Y(NPY)及促性腺激素释放激素(GnRH)在下丘脑弓状核...     

Kisspeptin/kiss1r系统在多囊卵巢综合征大鼠卵巢中的表达及其对卵泡发育障碍的可能作用机制

目的:探讨多囊卵巢综合征(PCOS)大鼠卵巢中kisspeptin/kisslr系统的表达及其对PCOS大鼠卵泡发育障碍的可能作用机制。方法:构建PCOS大鼠模型;免疫组织化学检测实验大鼠卵巢中kisspeptin/kiss1r的表达;免疫印迹和qRTPCR分别检测kisspeptin/kisslr蛋白和mRNA水平。结果:与正常组相比,PCOS组的体质量及卵巢质量明显增加,血清雄激素、黄体生成素水平明显增加,卵泡刺激素水平变化无统计学差异。PCOS大鼠卵巢中含有较多发育早期的小卵泡及闭锁卵泡,囊状扩张卵泡明显增加,颗粒细胞层数减少至2~3层,黄体数量明显下降;kisspeptin/kisslr蛋白和mRNA水平明显降低。结论:卵巢表达的kisspeptin/kisslr在PCOS大鼠卵泡发育障碍中可能通过调节颗粒细胞发挥作用,本研究将为进一步探讨PCOS的发病机制提供一定的理论基础。     

电针上调食源性肥胖致不孕大鼠下丘脑弓状核内GnRH的表达

目的 探讨电针对食源性肥胖致不孕大鼠中枢性作用中,能否上调下丘脑弓状核GnRH的表达.方法 将100只刚断乳雌性SD大鼠随机分为2组:①正常组20只,喂以普通饲料;②高能饲料组80只,给予高能饲料,筛选出肥胖不孕大鼠22只,并将其随机分成两组,即对照组和电针组.检测血清雌二醇(E2)、睾酮(T)、瘦素(leptin) ...     

GPR54/kisspeptin的生殖内分泌作用研究进展

GPR54是新发现的视黄酸家族G蛋白偶联受体,其内源性配体为k isspeptin。GPR54/K iss-1 mRNA分布于中枢神经系统的下丘脑、弓状核等多个区域,其表达随大鼠不同生理阶段及性周期的改变而变化。GPR54/k isspeptin在调节促性腺激素LH和FSH的释放,促进生殖器官的发育及青春期启动中具有相当重要的作用。     

Kisspeptin/GPR54系统与促性腺轴及青春期发育关系的研究进展

1青春期发育的研究现状 下丘脑合成和分泌的促性腺激素释放激素（gonadotropin-releasing hormone，GnRH）是启动下丘脑-垂体-性腺（hypothalamic-pituitary-gonadal，HPG）轴的一种重要神经激素，以脉冲方式释放至垂体门脉循环，刺激垂体卵泡刺激素（FSH）和黄体生成素（LH）的合成和释放，     

不同时程应激对大鼠下丘脑弓状核和室周核神经细胞的影响

目的:观察不同时程应激对大鼠下丘脑弓状核和室周核神经细胞的影响及酪氨酸羟化酶(TH)的表达变化,为应激性损伤的机制研究提供病理形态学依据。方法:建立每日束缚固定8 h加冰水游泳5 min的应激大鼠模型,分为1、3、7、14、21d组及各时间点正常对照组,采用硫堇染色观察弓状核、室周核内神经细胞尼氏体变化及细胞形态学改变;TH免疫组织化学标记观察大鼠下丘脑弓状核和室周核内多巴胺能神经细胞阳性表达变化,采用全景组织细胞定量分析系统,进行统计分析。结果:较长时程反复的应激刺激导致了大鼠弓状核和室周核神经细胞细胞质内尼氏体消失及部分细胞固缩。TH免疫组织化学标记显示随着应激时程的延长,下丘脑弓状核和室周核内多巴胺能神经细胞阳性表达数目减少。结论:较长时程的反复应激刺激可导致大鼠下丘脑弓状核和室周核神经细胞损伤及多巴胺能神经细胞缺失。     

5/6肾切除大鼠血清瘦素水平变化及其与肾小球硬化关系

目的:观察5/6肾切除大鼠血清瘦素(leptin)水平及其与肾小球硬化指数(GSI)、肾小球局部转化生长因子-β1(TGF-β1)表达、细胞外基质(ECM)的关系。方法:选用SD雄性大鼠14只,其中8只通过5/6肾切除法制造慢性功能衰竭模型,另6只为假手术组作为正常对照。术后第6周末各组大鼠进行血清肌酐(Scr)、尿素氮(BUN)及leptin的测定,处死大鼠,取出肾组织进行病理组织形态学观察,并采用免疫组织化学方法检测肾小球TGF-β1、Ⅳ型胶原(ColⅣ)及纤维连接蛋白(FN)表达。并对血清leptin水平与GSI、TGF-β1、ColⅣ及FN之间的关系进行相关性分析。结果:5/6肾切除大鼠血清瘦素(leptin)水平显著高于假手术组(14.88±1.46ng/mlvs10.84±2.67ng/ml,P<0.01),并与GSI、TGF-β1、ColⅣ及FN呈显著正相关(P<0.01)。结论:高瘦素血症可能是引起肾小球硬化的机理之一。     

Introduction: Listening to the Voice of the C/S/X: Consumer/Survivor/Expatient

People who are receiving services for professionally diagnosed psychological disabilities often are not consulted about the nature of those services, or their willingness to participate in them. This issue of the journal presents the autobiographical accounts of four such people, followed by commentaries about those accounts by three professional service-givers. This collection emphasizes the need to obtain informed consent for any psychological services that are offered, for ethical, humane, and professional reasons.     

Sequence of the nucleoprotein gene of influenza A/parrot/Ulster/73

The nucleotide sequence of the nucleoprotein (NP) gene of the avian influenza A virus strain A/parrot/Ulster/73 (H7N1) has been determined. The gene (RNA segment 5) consists of 1565 bases. The only large open reading frame of the complementary RNA codes for a protein of 498 amino acids. A comparison of its sequence with that of three other influenza virus NPs shows that the NP of the parrot Ulster strain, although closely related to the NP of the other avian strain (A/FPV/Rostock/34), is definitely more closely related genetically to the NPs of the two human influenza strains, A/PR/8/34 and A/NT/60/68 than that of FPV. This raises the question how far the NP gene can cross the species barrier by reassortment and become adapted by mutation to the new host.     

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.     

Evaluation of the A/Seal/Mass/1/80 virus in squirrel monkeys

An influenza A virus isolated from seals [A/Seal/Mass/1/80 (H7N7)] and an isolate of this virus obtained from a human conjunctiva were evaluated for replication and virulence in squirrel monkeys. When the seal virus was administered intratracheally, it replicated in lungs and nasopharynges and induced illness almost to the same extent that a human influenza A virus [A/Udorn/72 (H3N2)] did. In one monkey that died of pneumonia, the seal virus was recovered from spleen, liver, and muscle as well as lung. After conjunctival administration in monkeys, the seal virus replicated to a peak titer in the conjunctivae 30-fold greater than that attained by the human virus, but this difference was not statistically significant. In contrast, the seal virus replicated less well than the human virus in the tracheae and nasopharynges when administered by the conjunctival route. These results indicate that the seal virus can replicate efficiently in primates, that it can spread systemically, and that it might differ from human virus in being able to replicate slightly better in primate conjunctival tissue.     

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.     

Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway

The VP40 matrix protein of Ebola virus is able to bud from mammalian cells as a virus-like particle (VLP). Interactions between L-domain motifs of VP40 and host proteins such as Tsg101 and Nedd4 serve to facilitate budding of VP40 VLPs. Since intracellular levels of calcium are known to influence localization and function of host proteins involved in virus budding, we sought to determine, whether alterations of calcium or calmodulin levels in cells would affect budding of VP40 VLPs. VP40 VLP release was assessed in cells treated with BAPTA/AM, a calcium ion chelator, or with ionomycin, a calcium ionophore. In addition, VLP budding was assessed in cells treated with W7, W13, or TFP; all calmodulin antagonists. Results from these experiments indicated that: (i) budding of VP40 VLPs was reduced in a dose-dependent manner in the presence of BAPTA/AM, and slightly enhanced in the presence of ionomycin, (ii) VP40 VLP budding was reduced in a dose-dependent manner in the presence of W7, whereas VP40 VLP budding was unaffected in the presence of cyclosporine-A, (iii) budding of VSV-WT and a VSV recombinant (M40 virus) possessing the L-domains of Ebola VP40 was inhibited in the presence of W7, W13, or TFP, (iv) inhibition of virus budding by W7, W13, and TFP appears to be L-domain independent, and (v) the mechanism of calcium/calmodulin-mediated inhibition of Ebola VLP budding may involve the Ras/Raf/MEK/ERK signaling pathway.     

Sequence of the hemagglutinin gene from influenza virus A/Seal/Mass/1/80

A double-strand DNA copy of the influenza virus A/Seal/Mass/1/80 (H7N7) [seal] hemagglutinin (HA) gene was cloned into the plasmid pAT153/PvuII/8 and sequenced to deduce the primary amino acid sequence. The gene is 1731 nucleotides long and codes for a protein of 560 amino acids with a nonglycosylated molecular weight of 62098 Da. The deduced amino acid sequence displays similarities to all other sequenced hemagglutinins by retaining six of seven potential glycosylation sites, showing conversation in the number and position of cysteine residues, conservation in the fusion and anchor peptides, and conservation in the putative receptor site of the molecule. However, three features of the primary amino acid sequence could be distinguished from the H7 amino acid sequence of A/fowl plague/Rostock/34 (FPV), another avian H7 influenza virus which does not produce disease in mammals. First, the seal HA sequence has three fewer amino acids in the connecting peptide region of the HA than FPV. This lack of multiple basic amino acids in the connecting peptide is similar to that found in avirulent H7 avian strains and to mammalian serotypes H1, H2, and H3. Second, the seal HA has gained four additional proline residues, all in HA1, as compared to FPV. These residues may alter the tertiary structure of the HA and ultimately contribute to the biological features of this virus. Third, the seal HA has lost a potential carbohydrate attachment site at residue 149 which lies at the tip of the HA structure. The loss of this carbohydrate could alter the seal HAs interaction with host cell receptors.     

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.     

Localisation of the temperature-sensitive defect in the nucleoprotein of an influenza A/FPV/Rostock/34 virus

The nucleotide sequences of the nucleoprotein (NP) genes of fowl plague virus (FPV) and of a temperature-sensitive (ts) mutant (ts81) derived therefrom have been determined. The ts81-NP nucleotide sequence possesses a single nucleotide substitution in comparison to the wild type. This causes an amino acid exchange at position 332 of the NP. An alanine in the wild type-NP is substituted by a threonine in ts81-NP. This substitution leads to a significant difference in the secondary structure prediction. Although this mutation is located within the karyophilic region of the NP, the accumulation of the NP in ts81-infected cells is not significantly affected at 40 degrees C. Therefore, we assume that the cooperation with one of the polymerase proteins (P) is interfered with at 40 degrees C, leading to the loss of viral vRNA or replicative cRNA synthesis. The comparison of the FPV-NP nucleotide sequence to a previously published sequence of the same strain (Tomley and Roditi, 1984) highlights ten nucleotide differences, four of them leading to amino acid substitutions.