卵巢内瘦素受体缺失对卵巢生殖功能和全身糖脂代谢的影响

为探索卵巢内瘦素信号传导缺失对卵巢自身功能和全身糖脂代谢的影响,阐明瘦素对外周生殖系统的直接作用,进行了以下研究:①对瘦素长受体缺失(LR-)的雌性B6.Cg-m / Leprdb小鼠和其同窝出生的有完整LR的雌性野生型C57BU6J小鼠(LR ),在性成熟期后的12周,进行卵巢相互移植,构建实验小鼠的LR基因型组合分别为A(躯体 ,卵巢-)、B[躯体 ,卵巢-)、C(躯体 ,卵巢 )、D(躯体-,卵巢 )、E(躯体-,卵巢-)共5组(n=5),移植后观测2个动情周期取材:②检测各自的全身糖脂代谢指标、卵巢周期和生殖激素;③卵泡刺激素(0.75 IU/g)刺激试验后,用免疫组化和蛋白质印迹(WB)方法,检测卵巢内脂代谢信号蛋白包括Janus激酶(JAK2)、磷酸化的丝裂原激活蛋白激酶(p-ERK)和低密度脂蛋白受体(LDLR)的表达.结果显示:①D、E组与A、B、C 3组比较,体重相差约1倍,性腺周围脂肪垫重量相差约10倍(P<0.01);血葡萄糖、低密度脂蛋白胆固醇(LDL-C)和胰岛素水平相差2-3倍(P<0.01);A、B、C 3组的全身血糖脂指标相似,D和E 2组的高糖脂血症升高程度也相似.(②A、B、C 3组小鼠均恢复正常的动情周期,而D、E 2组则始终处于动情间期;小鼠16周龄时D、E组与A、B、C 3组比较:卵巢重量及E2、P、FSH、LH水平显著下降(P<0.05 J.③瘦素刺激后JAK2在A、B、D和E组的卵巢中表达均呈弱阳性,而C组卵巢内呈强阳性表达;FSH刺激后p-ERK和LDLR在A、B、C 3组卵巢内均呈强阳性表达,而D和E组则表达下降.进而得出结论:①单纯卵巢内瘦素信号缺失并不影响卵巢自身的生殖功能、脂质代谢和全身的糖脂代谢.②瘦素受体全身缺失小鼠的卵巢功能障碍,与卵巢外的全身因素即高糖脂血症和低促性腺激素状态有关.③高糖脂血症降调节卵巢内的瘦素信号蛋白JAK2的表达,诱导小鼠卵巢脂质化和功能障碍;FSH刺激可诱导ERK磷酸化后LDLR的表达增强,缓解小鼠卵巢的脂质化和功能障碍.     

补肾活血颗粒对多囊卵巢综合征大鼠血清性激素水平、 血糖及胰岛素的影响

目的 观察补肾活血颗粒对多囊卵巢综合征（PCOS）模型大鼠血清性激素水平、血糖及胰岛素的影响。方法 采用颈背部皮下注射脱氢表雄酮（DHEA）造模法建立PCOS大鼠模型,将72只雌性SD大鼠随机分为正常对照组、模型组、二甲双胍组、中药高剂量组、中药中剂量组、中药低剂量组,每组12只,各组给予相应药物连续灌胃 20 d,用酶联免疫吸附测定法测定血清17-羟孕酮（17-OHP）、促黄体生成素（LH）、睾酮（T）、促卵泡素（FSH）、空腹 血糖（FPG）及胰岛素（INS）水平。结果模型组血清中17-OHP、LH、T、FSH、FPG、INS水平明显高于正常对照组 (P<0.01 ,P<0. 05 ),中药高剂量组降低大鼠血清LH、T、FSH及INS水平最为明显(P<0.01),二甲双胍组、中药中剂量组显著降低FPG（P<0. 01）,中药各治疗组均能降低17-OHP（P<0. 05）。结论 补肾活血颗粒能对PCOS 大鼠有一定的治疗作用,可改善激素水平、血糖及胰岛素的异常     

卵巢局部瘦素受体缺失对卵巢生殖功能影响的分子机制

为探索卵巢局部瘦素信号缺失对卵巢自身功能的影响,进行以下研究:①对瘦素长受体(LR)缺失(LR-)的雌性B6.Cg-m+/+Leprdb小鼠及其同窝出生的有完整LR的雌性野生型C57BL/6J小鼠(LR+)进行卵巢交互移植,在同一雌性野生型C57BL/6J小鼠体内构建两侧卵巢不同的LR基因型,分别标记为KO(卵巢LR-)、WT(卵巢LR+)两组.②监测卵巢的周期变化;动情周期恢复后各组均予GnRH-a降调节后取材,RT-PCR检测分子生物学指标.结果显示:①卵巢移植术后小鼠仍有正常的动情周期,经GnRH-a降调节后,小鼠失去动情周期的变化;②RT-PCR结果显示,KO组卵巢中Star、Cyp17、Cyp19、Jak2、Star3、Pias3 mR-NA的表达量均明显低于、WT组,Ob-Rb mRNA在KO组卵巢中无表达,WT组有表达,Socs3 mRNA在两组间表达无明显差异.研究表明,卵巢内存在完整的瘦素信号传导通路;瘦素受体缺失及外周糖脂代谢环境对卵巢功能的影响并不重要,重要的是下丘脑-垂体对卵巢的调控作用,其对卵巢作用的分子机制是调控卵巢甾体激素合成酶基因的表达.     

孕期高雄大鼠子代实验性多囊卵巢综合征模型构建

通过母体高雄环境建立子代实验性多囊卵巢综合征(PCOS)大鼠模型,并证明此模型大鼠存在卵巢局部胰岛素抵抗。方法是给孕16d的雌鼠皮下注射丙酸睾丸酮,诱导其子代雌鼠发生PCOS,观察子代大鼠体重、动情周期变化、性激素变化及糖代谢变化、卵巢重量和卵巢形态变化、肌肉组织蛋白印迹测定PI-3K和MAPK通路关键蛋白的表达、卵巢免疫组化和蛋白印迹测定PI-3K和MAPK通路关键蛋白的表达确定。结果显示:①动情周期失去规律变化,提示无排卵;②血清17-OHP,A2,T升高;③卵巢组织学检查可镜下看到早期发育的较多的小卵泡、闭锁卵泡,囊状扩张卵泡明显增加,颗粒细胞层数减少,黄体数量明显减少;④OGTT:1h血糖及胰岛素高于对照组,模型组存在着胰岛素抵抗;⑤卵巢局部PI-3K和MAPK途径关键蛋白有表达;⑥卵巢局部蛋白印迹测定PI-3K和MAPK途径关键蛋白IRS-1,P85,GLUT4降低,ERK1升高。由此表明:①此模型大鼠符合PCOS的诊断标准;②此模型大鼠存在卵巢局部胰岛素抵抗。     

大豆异黄酮对卵巢切除大鼠糖脂代谢和血清瘦素、抵抗素表达的影响

为探讨大豆异黄酮对卵巢切除大鼠糖脂代谢和血清瘦素、抵抗素表达的影响,将12周SD大鼠分成3组,假手术(Sham)组、卵巢切除(OVX)组和大豆异黄酮(OVX+Gen)组,每组7只,卵巢切除术后10 d,OVX组和Sham组大鼠每天腹腔注射生理盐水0.1 ml/只,OVX+Gen组每天注射大豆异黄酮0.2 mg/只,连续注射30 d后处死取血,检测子宫病理改变、血糖血脂和胰岛素水平、血清瘦素和抵抗素含量.结果发现卵巢切除后大鼠总胆固醇、HDL和LDL均增加(P<0.05),但胰岛素、血糖、甘油三酯、血清瘦素和抵抗素水平变化不明显(P>0.05);卵巢切除后补充大豆异黄酮,可促进子宫内膜上皮部分增生,但胰岛素、血糖、总胆固醇、甘油三酯、LDL和HDL、血清瘦素和抵抗素水平均无明显变化(与OVX组相比,P>0.05),结果提示大豆异黄酮对卵巢切除大鼠糖脂代谢参数和血清瘦素、抵抗素水平影响不大.     

电针对多囊卵巢综合征模型系统的调控

通过母体高雄环境建立子代雌性大鼠高雄激素血症和胰岛素抵抗模型,探讨针刺对孕期高雄化大鼠的治疗效果以及针刺调节卵巢局部胰岛素信号传导关键分子磷脂酰肌醇3激酶(PI-3K)和丝裂原激活蛋白激酶(MAPK),明确针刺治疗胰岛素抵抗的分子机制。在Wistar雌性大鼠颈背部皮下注射丙酸睾丸酮,连续3d。以其所生雌鼠作为实验对象,观察其体重及动情周期,检测血清性激素,17-羟孕酮(17-OHP)、雄烯二酮(A2)及OGTT试验测血糖、胰岛素水平,然后给予针刺治疗,观察其对生殖与代谢功能的影响。运用免疫组化及蛋白印迹方法,检测骨骼肌和卵巢组织中胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)、磷脂酰肌醇3-激酶(PI-3K85)、葡萄糖转运蛋白-4(GLUT4)、细胞外信号激酶-1(ERK-1)和17羟化酶(CYP17)蛋白表达。结果显示:①模型鼠无排卵,高雄、糖耐量减低,胰岛素抵抗;②针刺可以恢复动情周期,降低体重,降低LH、17-OHP、A2、T水平,升高E2水平;降低OGTT试验1h血糖水平,降低空腹和1h胰岛素水平,降低HOMA指数;③骨骼肌和卵巢局部产生胰岛素抵抗后,相应的IRS-1、IRS-2、PI-3K85和GLUT4的蛋白表达下降,ERK-1、CYP17的表达升高;针刺能逆转蛋白的异常表达。由此得出以下结论:①孕期注射丙酸睾丸酮,所生子代雌鼠有生殖和代谢异常,符合PCOS患者临床特征;②针刺能够降低雄激素,降低血糖,提高胰岛素敏感性,降低体重;③针刺能够调节模型动物骨骼肌和卵巢局部胰岛素信号传导蛋白表达和卵巢局部雄激素合成酶表达。     

绒毛膜促性腺激素刺激对多囊卵巢综合征患者血管活性因子的影响

探讨促排卵治疗对多囊卵巢综合症(PCOS)患者血管活性因子血管紧张素-Ⅱ(AT-Ⅱ)、白介素-6(IL-6)、胰岛素样生长因子-1(IGF-1)的影响.21例PCOS患者与8例正常女性在自然或人工月经周期的第2 d卵泡期进行绒毛膜促性腺激素(HCG)促排卵,于试验前及试验后3、6、12、18、24 h静脉取血,放免法测定血清AT-ⅡI、L-6I、GF-1浓度.HCG刺激后PCOS组的血清AI-Ⅱ水平在各取血时点均升高,但只有3 h取血点是显著升高(P<0.05).PCOS组HCG刺激后各时点的血清AI-Ⅱ水平均高于对照组,也只有3 h取血点是显著高于对照组.血清IL-6及IGF-1水平在试验前后各时间点间差异均无显著性.结果提示HCG刺激促进PCOS患者外周血AT-Ⅱ水平升高.     

长期有氧运动对肥胖大鼠血液及肝脏糖脂代谢的影响

将15月龄SD雄性大鼠分为正常对照组(CON)、高脂饮食组(HFD)和高脂饮食运动组(HFD+CE)。实验结束处死后取材,测定各组大鼠空腹血糖(PBG)、血清甘油三酯(TG)、胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)、肝脏TG、游离脂肪酸(FFA)及肝糖原(LG)浓度,空腹胰岛素水平(FINS)及胰岛素抵抗指数(HOMA-IR)。结果显示:与CON组相比,HFD组大鼠HDL-C、LG显著降低(P<0.01,P<0.05),PBG无显著性变化(P>0.05),TG、FINS、HOMA-IR均显著升高(P<0.05),血清TC、LDL-C、L-TG、L-FFA升高极显著(P<0.01);与HFD组相比,HFD+CE组大鼠HDL-C升高极显著(P<0.01),TC、PBG无显著变化(P>0.05),LG、L-TG、FINS、HOMA-IR均显著下降(P<0.05),LDL-C、L-FFA降低极显著(P<0.01)。结论:长期有氧运动对高脂诱导的肥胖大鼠具有明显降脂作用,并能降低血清胰岛素水平,改善胰岛素抵抗和肝脏糖脂代谢紊乱。     

长期心理应激对卵巢功能影响的实验研究

目的 研究长期心理应激对卵巢功能的影响.方法 应用束缚的方法产生心理应激,用酶联免疫法测定心理应激BALB/C雌小鼠CORT(皮质酮),用放射免疫法测E2(雌二醇)、P(孕酮);同时观察卵巢形态、各级卵泡数及妊娠率和产子数.结果 长期心理应激能使雌性小鼠CORT含量升高(P<0.05);血清E2、P的含量降低(P<0.01);卵巢形态学发生明显变化,窦前卵泡及窦卵泡数量减少(P<0.05);对小鼠妊娠率无明显影响(P>0.05),但产仔数减少,有显著差异(P<0.05).结论 长期心理应激可影响卵巢功能.     

骨髓间充质干细胞对损伤卵巢的修复作用

目的:探讨骨髓间充质干细胞移植对损伤卵巢的修复作用。方法:通过贴壁筛选法培养、纯化GFP转基因小鼠MSCs。将健康的雌性ICR小鼠(6 w)随机分为4组:正常对照组(A组,n=6)、MSCs移植组(B组,n=6)、MSCs治疗组(C组,n=6)、损伤组(D组,n=6)。B组腹腔移植MSCs,C、D组小鼠卵巢在无菌条件下,用体积分数为10%H2O2损伤卵巢建立损伤模型,24 h后C组腹腔移植MSCs,D组注射等体积的PBS。各组每周取卵巢,采用外观拍照、PCR检测GFP基因和病理切片观察MSCs是否对损伤卵巢有修复作用。结果:PCR结果表明MSCs经腹腔移植可到达正常卵巢,也可到达受损伤卵巢;C组移植MSCs后小鼠卵巢恢复的比D组快。结论:MSCs通过腹腔移植可到达卵巢,并可促进损伤卵巢的修复。     

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.     

Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.     

BALB/c突变无毛小鼠免疫学特性指标的研究

BALB c突变无毛小鼠的突变基因是位于小鼠第 11号染色体上 ,定位结果表明该突变为一种新的影响小鼠皮肤和被毛结构的突变 ,被命名Uncv(Uncovered) ,已得到小鼠遗传国际命名委员会的认可 ,并已被小鼠基因组资料库收录。皮肤是机体最大的器官 ,也是机体与外界的主要生理屏障 ,它具有产生、维持局部免疫应答和炎症反应的能力 ,许多免疫应答都发生于皮肤。小鼠皮肤和被毛结构的突变是否影响机体的免疫功能 ,解剖观察和饲养繁殖情况表明该小鼠免疫器官健全 ,在普通饲养环境中能够繁殖。为了进一步研究BALB c突变无毛小鼠的突变基因免疫功能 ,…     

Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells

Specific genes can be inactivated or mutated in the mouse germ line. The phenotypic consequences of the mutation can provide pivotal information on the function of the gene in development and maintenance of the mammalian organism. The procedure entails homologous recombination in embryonic stem cells, which, on fusion to recipient blastocysts, give rise to chimaeric mice that can transmit the mutant gene to their offspring. Inbreeding can then yield mice carrying the mutation in both alleles allowing the phenotypic analysis of recessive mutations. In addition to mice lacking a particular gene function, cell lines carrying null alleles of normally expressed genes can be instrumental in assessing the function of the gene. These cell lines can either be obtained from homozygous animals or, should the mutation be lethal early in embryonic development, be generated by consecutive inactivation of both alleles by homologous recombination in cultured cells. Here we illustrate the feasibility of this latter approach by the efficient consecutive inactivation of both alleles of the pim-1 proto-oncogene in embryonic stem cells.     

Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice

Reactive aldehydes are common carcinogens. They are also by-products of several metabolic pathways and, without enzymatic catabolism, may accumulate and cause DNA damage. Ethanol, which is metabolised to acetaldehyde, is both carcinogenic and teratogenic in humans. Here we find that the Fanconi anaemia DNA repair pathway counteracts acetaldehyde-induced genotoxicity in mice. Our results show that the acetaldehyde-catabolising enzyme Aldh2 is essential for the development of Fancd2(-/-) embryos. Nevertheless, acetaldehyde-catabolism-competent mothers (Aldh2(+/-)) can support the development of double-mutant (Aldh2(-/-)Fancd2(-/-)) mice. However, these embryos are unusually sensitive to ethanol exposure in utero, and ethanol consumption by postnatal double-deficient mice rapidly precipitates bone marrow failure. Lastly, Aldh2(-/-)Fancd2(-/-) mice spontaneously develop acute leukaemia. Acetaldehyde-mediated DNA damage may critically contribute to the genesis of fetal alcohol syndrome in fetuses, as well as to abnormal development, haematopoietic failure and cancer predisposition in Fanconi anaemia patients.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth

Oestrogen exerts its influence on target organs through activating oestrogen receptors (ERs) and regulating downstream genes by means of their oestrogen-responsive elements. Efp, a target gene product of ER alpha, is a member of the RING-finger B-box coiled-coil (RBCC) motif family. Efp is predominantly expressed in various female organs as well as in breast cancers, and is thought to be essential for oestrogen-dependent cell proliferation and organ development Efp-disrupted mice display underdeveloped uteri and reduced oestrogen responsiveness. Here we show that Efp is a RING-finger-dependent ubiquitin ligase (E3) that targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes G2 arrest. We demonstrate that tumour growth of breast cancer MCF7 cells implanted in female athymic mice is reduced by treatment with antisense Efp oligonucleotide. Efp-overexpressing MCF7 cells in ovariectomized athymic mice generate tumours in the absence of oestrogen. Loss of Efp function in mouse embryonic fibroblasts results in an accumulation of 14-3-3 sigma, which is responsible for reduced cell growth. These data provide an insight into the cell-cycle machinery and tumorigenesis of breast cancer by identifying 14-3-3 sigma as a target for proteolysis by Efp, leading to cell proliferation.