海带多糖对高脂血症小鼠血清瘦素水平及瘦素受体表达的影响

本实验目的在于探讨海带多糖对实验性高脂血症小鼠瘦素及其受体水平的影响及其可能的机制。将健康雄性昆明小鼠80只,随机分为正常饲料组和高脂饲料组,后组以高脂饲料喂养建立高脂血症动物模型。将建模成功的小鼠随机均衡分为高脂模型组和海带多糖治疗组,后组以海带多糖灌胃干预。自动生化分析仪检测小鼠空腹血脂水平,ELISA测定血清瘦素(Leptin)含量,免疫组织化学法和Western blot检测下丘脑组织中瘦素受体(Lep-R,OBR)蛋白表达,RT-PCR检测OBR mRNA表达。结果示海带多糖治疗组小鼠血脂及血清Leptin水平较模型组显著降低(P0.05),下丘脑OBR mRNA及其蛋白表达水平较模型组显著升高(P0.05)。说明海带多糖可以通过提高下丘脑OBR的水平,改善瘦素抵抗,从而发挥降脂作用。     

间歇性低氧对肥胖小鼠瘦素及其受体表达的影响

为探讨适度低氧环境对体重的影响及其作用机制,明确瘦素在其中的作用,用高脂饮食建立小鼠肥胖模型并观察间歇性低氧的干预效果。健康昆明小鼠随机分为4组（每组20只）,正常对照组：喂正常食物,不进行间歇性低氧训练;低氧组：喂正常食物,并进行间歇性低氧训练;肥胖组：喂高脂、高糖食物,但不进行间歇性低氧训练;低氧＋肥胖组,喂高脂、高糖食物,并进行间歇性低氧训练。40d后,测量小鼠体重,用酶联免疫吸附法测定血清瘦素水平,免疫组织化学检测肝脏瘦素受体表达,苏丹Ⅲ染色检测肝脏脂肪细胞分布和密度。结果显示,与正常对照组相比,肥胖组小鼠平均体重和平均血清瘦素水平显著升高,肝脏分布大量脂肪细胞,提示高脂模型建立成功;经过间歇性低氧训练后,低氧组和低氧＋肥胖组小鼠的平均体重及肝脏脂肪细胞分布密度和范围分别较对照组和肥胖组低,而血清瘦素水平明显增高;低氧＋肥胖组小鼠肝脏瘦素受体的表达高于肥胖组。结果提示,适度的间歇性低氧可以通过提高血清瘦素水平和增强肝脏瘦素受体表达而使体重减轻,并有效防止肝细胞脂肪变。     

瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达的影响

目的研究瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体(Ob-R)mRNA表达的影响。方法采用六孔板培养皿培养鼠平滑肌细胞,5个孔作为一个浓度组,通过半定量的RT-PCR方法,分别观察了不同浓度的大鼠瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达水平的影响。结果通过半定量RT-PCR,我们检测了短型瘦素受体(Ob-Ra)和长型瘦素受体(Ob-Rb)的mRNA表达水平,瘦素受体(Ob-R)mRNA表达水平随着瘦素及IL-6浓度增加而上升,随着胰岛素的浓度增加而下降。结论瘦素及IL-6可在体外上调平滑肌细胞瘦素受体的表达,胰岛素可下调平滑肌细胞瘦素受体的表达。这三个因子参与了瘦素受体的表达调控,对我们了解瘦素抵抗的发生机制有重要意义。     

味觉刺激后瘦素及其受体表达的变化

目的 :研究经口腔味觉刺激后 ,大鼠血清瘦素水平和脑瘦素受体表达情况。方法 :给大鼠口腔味觉刺激 ,味觉刺激物包括 3mol/L蔗糖 ,5mmol/L糖精钠 ,0 .1mol/LNaCl,0 .0 1mol/LHCl,1mmol/L奎宁H2 SO4和 0 .1mol/L谷氨酸钠 ,采用大鼠瘦素放免试剂盒测定血清瘦素水平。应用免疫组织化学ABC法对大脑切片进行染色 ,一抗为特异性羊抗瘦素受体IgG。 结果 :与对照组 (以蒸馏水代替味觉刺激物 )相比 ,仅甜味组 (包括蔗糖和糖精钠 )血清瘦素水平升高 (P <0 .0 5 )。杏仁核、下丘脑、臂旁核和孤束核等与味觉和摄食明显相关的核团均存在瘦素受体免疫反应 (LR IR)阳性细胞 ,但是阳性细胞数目在味觉刺激组和对照组间无显著性差异。结论 :给大鼠甜味觉刺激后 ,血清瘦素水平升高。并且杏仁核这个在摄食的发动和引导中起重要作用的核团存在LR IR阳性细胞。这些结果提示瘦素可能通过调节味觉感受而影响摄食 ,有必要对瘦素在味觉感受方面的作用进一步研究     

瘦素受体的研究进展

瘦素的体重调节作用是通过瘦素受体介导的。随着分子生物学技术的发展,已以受体的基因表达、结构、功能等方面取得了一些进展。肥胖患者常合并高胰岛素血症、胰岛素抵抗和瘦素抵抗,说明瘦素受体与肥胖及其相关疾病有着不可分割的联系。     

运动对高脂膳食雌鼠性发育和瘦素受体mRNA表达的影响

目的：探讨运动干预对高脂膳食雌性大鼠肥胖与性发育不良的影响及其作用的机制。方法：雌性Wister大鼠,按体重随机分为正常对照组（C组）、高脂膳食对照组（HC组）、高脂膳食运动组（HE组）,采用无负重递增负荷游泳训练8周,观察其肥胖、性发育与脂肪瘦素受体的关系。结果：8周的高脂饮食导致大鼠肥胖,卵巢重量、血清雌二醇、瘦素水平明显升高（P〈0．01,P〈0．05,P〈0．05）,脂肪的Leptin mRNA和体长显著下降（P〈0．01）;8周的游泳训练使卵巢重量、血清雌二醇水平和瘦素水平明显下降（P〈0．05;P〈0．01）,脂肪Leptin mRNA表达和体长显著上升（P〈0．01）;且血清雌二醇与肥胖度各指标呈正相关,与体长呈负相关;血清瘦素与肥胖度各指标和反映性发育水平各指标均呈正相关。结论：①高脂膳食可诱导雌性大鼠肥胖,使血瘦素水平升高,瘦素受体的基因表达下调。出现了瘦素抵抗,性发育增强。②耐力训练可明显降低体脂和血瘦素水平,上调脂肪的瘦素受体的基因表达,有效的缓解了瘦素抵抗和性发育增强。     

瘦素对骨重建的神经调节

骨重建是骨骼保持自我更新的过程,以往认为骨重建的调节仅涉及骨骼局部的自分泌、旁分泌等方式。近年来的研究发现,中枢神经系统对骨重建的维持发挥关键作用,瘦素是其中重要的调控因子。本文简要综述瘦素通过交感神经系统、神经肽(可卡因安非他明调节转录肽,CART),以及神经介素U对骨重建的调节作用及其分子基础。     

瘦素及其受体在宫颈癌中的表达和意义

目的探讨瘦素(leptin)及其受体(OB-R)在宫颈癌中的表达与临床病理参数的关系。方法采用免疫组织化学技术检测10例慢性宫颈炎、12例宫颈上皮内瘤样病变、50例宫颈癌中leptin和OB-R的表达,利用CD34标记宫颈癌血管内皮细胞并计算微血管密度(MVD)。结果leptin和OB-R仅在10例慢性宫颈炎、12例上皮内瘤样病变的鳞状上皮层中呈弱阳性表达;50例宫颈癌中Leptin染色( )39例,OB-R染色( )33例;宫颈癌中leptin表达、MVD与肿瘤分化程度、浸润深度和是否远处转移呈正相关(P<0.05),与患者年龄和肿瘤组织学分型无关(P>0.05);OB-R表达与浸润深度和是否远处转移呈正相关(P<0.05),与患者年龄、肿瘤分化程度和组织学分型无关(P>0.05);leptin或OB-R染色( )MVD明显高于染色(-)者。结论Leptin是宫颈癌重要的血管生长因子,瘦素及其受体和MVD与肿瘤的恶性程度密切相关。     

大鼠局灶性脑皮质缺血/再灌注对脑神经元损伤作用及瘦素受体表达的改变

目的:研究脑缺血/再灌注(I/R)损伤后瘦素受体(OB-R)表达的变化情况.方法:雄性成年Wistar大鼠20只,随机分成4组:假手术24 h、72 h对照组及I/R 24 h、72 h实验组.线栓法制备大鼠局灶性脑皮质I/R损伤模型,在脑I/R后相应时间点分别处死大鼠,采用免疫组织化学、免疫电镜方法观察大脑皮质OB-R的表达,在光镜及电镜下观察神经元损伤改变.结果:左顶叶皮质锥体细胞、血管内皮、脉络丛发现有OB-R阳性表达;与假手术对照组相比,I/R 24 h(I/R早期)锥体细胞OB-R免疫反应阳性细胞表达减少(P＜0.05),I/R 72 h(I/R晚期)锥体细胞OB-R免疫反应阳性细胞减少更明显(P＜0.001);光镜及电镜对缺血中心区神经元的观察均显示I/R晚期的神经元损伤明显重于早期.结论:脑I/R损伤后早期神经元损害和迟发性神经元损害均伴随有OB-R的表达减少,且迟发性神经元损害表达减少更明显,因此在脑梗塞的防治中有必要对瘦素及其OB-R的作用进一步研究.     

非酒精性脂肪性肝病大鼠肝组织瘦素受体表达的变化

目的检测高脂饮食诱发的非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)大鼠肝组织瘦素受体的表达,探讨NAFLD瘦素抵抗的发生机制及瘦素抵抗在NAFLD发病中的作用.方法采用高脂饮食制备Wistar大鼠NAFLD模型;ELISA法测定大鼠血清瘦素(leptin,LP)浓度;全自动生化分析仪测血清甘油三酯(triglyceride,TG)、空腹血糖(fasting blood glucose,FBG),比色法测游离脂肪酸(free fatty acid,FFA),放射免疫法测空腹血清胰岛素(fasting insulin,FINS),计算空腹状态下胰岛素抵抗指数;对肝组织分别进行HE染色、苏丹Ⅳ染色、瘦素受体免疫组化染色,并进行半定量分析.利用SAS 8.0统计软件处理实验数据.结果模型组大鼠肝组织瘦素受体表达比正常组明显减弱,且与血清瘦素浓度、游离脂肪酸、胰岛素敏感指数、肝细胞脂变、炎症活动度显著负相关,相关系数分别为r = -0.83,-0.71, -0.65,-0.83, -0.87.多元线性回归分析显示,瘦素受体表达减弱是肝脂变的独立影响因素.结论肝组织瘦素受体表达减弱是NAFLD瘦素抵抗的重要病理机制之一,瘦素抵抗与胰岛素抵抗相互作用,共同促进了脂肪肝的发生发展.     

草鱼瘦素受体基因片段序列的克隆及其组织表达分析

根据斑马鱼、大西洋鲑和人等物种巴知的瘦素受体基因核苷酸保守区序列设计一对简并引物,通过RT-PCR法从草鱼肝胰脏中首次克隆获得草鱼瘦素受体基因的片段序列.该片段序列长713 bp,编码237个氨基酸,氨基酸序列分析表明草鱼瘦素受体基因片段氨基酸序列与其他物种的相似性在35％ -86％之间.通过邻接法(Neighbor Joining,NJ)构建系统进化树显示,鱼类的瘦素受体独立聚成一支,草鱼与金鱼、斑马鱼聚成一支,再与日本青鳉、黑点青鳉、红鳍东方鲀和大西洋鲑聚成一支.通过实时荧光定量PCR分析草鱼瘦素受体基因的组织差异表达,结果表明,草鱼瘦素受体基因在肝胰脏、肌肉、脑、心脏、脾和肠系膜脂肪组织中均有表达,其中在脾脏组织中表达量最多,显著高于其他组织(P＜0.05),其次是心脏、脑、肌肉和肠系膜脂肪组织,在肝胰脏组织中表达量最低,且显著低于其他组织(P＜0.05).     

雌激素和多巴胺对大鼠垂体组织中雌激素受体基因表达的影响

目的研究雌激素和多巴胺激动剂对雌激素受体在大鼠垂体组织表达的作用。方法20只成年雌性Wistar大鼠,切除卵巢后,随机分2组：（1）对照组（n=5）,皮下植入空白硅胶管;（2）雌激素组（n=15）皮下植入含有乙烯雌酚的硅胶管,8周后,两组各处死5只大鼠,雌激素组剩余大鼠（n=10）取出硅胶管,随机再分2组,安慰剂组（n=5）给予自来水灌胃,多巴胺组（n=5）给予溴隐亭（多巴胺激动剂）灌胃,用药4周后处死动物。放免法测定血清PRL水平,用反转录一聚合酶链反应（RT-PCR）方法检测ERs在各组垂体组织中的表达,以β-actin作为内参照,借助于计算机凝胶成像系统分析表达量。结果ERa,ERβ以及TERP在各组大鼠垂体组织均有表达,其中ERα和TERPmRNA水平在雌激素组明显高于对照组（P〈0．001）,在安慰剂组和多巴胺组的表达无明显差别。结论大鼠垂体组织中存在ER的表达,雌激素对ERα和TERP的表达具有升调节作用,多巴胺不影响雌激素受体的表达。     

Isoproterenol Decreases Leptin Expression in Adipose Tissue of Obese Humans

RICCI, MATTHEW R. AND SUSAN K. FRIED. Isoproterenol decreases leptin expression in adipose tissue of obese humans. Obes Res. Objective: We investigated the effects of the non-selective β-adrenergic agonist, isoproterenol (Iso), on leptin expression in human adipose tissue. Research Methods and Procedures: Subcutaneous (SQ) and omental adipose (OM) tissue taken during surgery from 12 morbidly obese subjects (10 women and 2 men) were cultured for up to 24 hours with insulin (7 nM) and/or dexamethasone (25 nM), a synthetic glucocorticoid, in the presence or absence of isoproterenol (10 μM). Adipose tissue was also acutely incubated for 3 hours in media alone with or without isoproterenol. Leptin secretion and leptin mRNA abundance were measured. Results: Iso acutely decreased leptin release by −30% (vs. no hormone controls) in fragments of OM and SQ adipose tissue. In 24-hour culture, addition of Iso (in the presence of insulin) resulted in lower leptin accumulation in the medium (−20–30%) and leptin mRNA levels (−40–50%) from both tissue depots. Culture with insulin and dexamethasone increased leptin expression vs. insulin alone. Addition of Iso with insulin and dexamethasone decreased media leptin (−40–60%) and leptin mRNA levels were lower (−65%) in Iso-treated adipose tissue from both depots after 24 hours. Iso effects were not detectable after 5 hours of culture. Discussion: We conclude that stimulation of β-adrenergic receptors may modulate leptin expression in human adipose tissue by two mechanisms: an acute effect on leptin release and a longer-term antagonism of stimulatory effects of insulin and dexamethasone on leptin mRNA expression. These mechanisms may contribute to the decline in serum leptin that occurs during fasting.     

西藏小型猪Leptin及其受体胞外区片段的克隆及原核表达

目的克隆及原核表达西藏小型猪瘦素（Leptin）成熟肽及瘦素受体胞外区片段。方法根据西藏小型猪瘦素序列（GenBank号：GQ240885.1）和猪瘦素受体基因胞外域序列（GenBank号：AF167719.1）分别设计并合成两对引物扩增瘦素、瘦素受体基因胞外域编码区1654-2319位片段,以西藏小型猪组织总RNA为模板,经反转录-聚合酶链反应（RT-PCR）方法获得了特异性片段。再以该两个特异性片段为模板,另外设计两对带有BanHⅠ和HidⅢ酶切位点的套式引物分别扩增瘦素64-504位（成熟肽编码区）和瘦素受体基因胞外域编码区1655-2314位的cDNA片段,将该两片段克隆入pMD18-T载体并转化感受态细菌E.coli DH5α测序并永久保存。此两片段经酶切后克隆到表达载体pRSET A的BamHⅠ和HindⅢ两酶切位点之间,构建重组质粒pR-OB和pR-OBR-a并在大肠杆菌E.coli BL21（DE3）中表达,SDS-PAGE电泳鉴定表达产物。结果在IPTG诱导下促使重组菌pR-OB表达了相对分子质量约18×103左右的融合蛋白;重组菌pR-OBR-a表达了相对分子质量约27×103左右的融合蛋白。结论说明重组质粒pR-OB、pR-OBR-a在大肠杆菌BL21（DE3）中分别可表达西藏小型猪瘦素成熟肽、瘦素受体片段蛋白,为进一步研究瘦素、瘦素受体功能和应用提供了基础。     

Expression and Biological Significance of Leptin,Leptin Receptor,VEGF, and CD34 in Colorectal Carcinoma

To investigate the expression and biological significance of Leptin, Leptin receptor, Vascular Endothelial Growth Factor (VEGF), and CD34 protein in colorectal carcinoma tissues. The expression of Leptin, Leptin receptor, VEGF, and CD34 was detected in 68 cases of colorectal carcinoma tissues, paired para-carcinoma tissues and normal colorectal tissues by Immunohistochemical SP Method. The results and related clinicopathological data were analyzed. The positive rate of Leptin, Leptin receptor, and VEGF was significantly higher in colorectal carcinoma tissues than that in paired para-carcinoma tissues and normal colorectal tissues. The expression of Leptin, Leptin receptor, and VEGF was correlated with grade of tumor differentiation, depth of bowel wall invasion, lymph node metastasis, Dukes stage, distant metastasis, and lympho/vascular tumor embolization. Microvessel density (MVD) value in colorectal carcinoma was significantly higher than that in para-carcinoma tissues and normal colorectal tissues, and the density in para-carcinoma tissues was higher than that in normal colorectal tissues. The expression of Leptin, Leptin receptor, VEGF, and MVD value in colorectal carcinoma was positively correlated. In conclusion, microvessel density value is an important index of the growth, invasion, and metastasis of colorectal carcinoma. The binding of Leptin and Leptin receptor promotes the proliferation of colorectal carcinoma cells. The synergy between Leptin and VEGF accelerates the angiogenesis in colorectal carcinoma and accelerates the invasion and metastasis of the tumor cells.     

胰岛素样生长因子在牙周组织工程中作用的研究进展

牙周膜细胞作为牙周组织工程中的重要种子细胞,在一定因素的诱导下,能够分化形成牙周组织的各种细胞,比如成纤维细胞,成骨细胞等,这些细胞能够分泌纤维蛋白,骨钙素等,进而钙化形成骨组织等与牙周组织相似或者相同的成分。胰岛素样生长因子作为重要的细胞因子,很多研究表明它在细胞迁移、增殖、分化、促进分泌等方面发挥作用,所以胰岛素样生长因子一直受到研究者的青睐。本文将对胰岛素样生长因子在牙周组织工程中的种子细胞的不同作用的研究进展进行综述,同时对牙周组织工程中的未来进行展望。     

Weight Loss Increases Soluble Leptin Receptor Levels and the Soluble Receptor Bound Fraction of Leptin

Objective: Soluble leptin receptor (sOB‐R) represents the main binding site for leptin in human blood. The aim of this study was to investigate the relationship between leptin and soluble leptin receptor and the bound/free ratio after pronounced weight reduction. Research Methods and Procedures: A total of 18 morbidly obese women participated in this prospective study. Subjects were examined for fat mass, leptin, and sOB‐R concentrations before and 1 year after Swedish adjustable gastric banding. Results: Anthropomorphic measures displayed a significant reduction of body mass index [(42.9 ± 5.6 to 32.9 ± 6.0 kg/m² (mean ± SD)]. Fat mass decreased from 56.3 ± 9.0 to 33.9 ± 12.5 kg. Plasma leptin concentration decreased from 44.6 ± 18.0 to 20.0 ± 13.1 ng/mL (p < 0.001), whereas the sOB‐R levels increased from 11.1 ± 3.6 to 16.6 ± 6.0 U/mL after weight‐reducing surgery. Thus, the sOB‐R bound fraction of leptin increased from 7% to 33%. Discussion: This work demonstrates a relationship between weight loss, leptin, and sOB‐R concentrations in vivo. During weight loss, leptin levels decreased, whereas sOB‐R levels and the receptor bound fraction of leptin increased. Thus, sOB‐R may negatively regulate free leptin.     

Kinetics of Leptin Binding to the Q223R Leptin Receptor

Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: k_a 1.76×10⁶±0.193×10⁶ M⁻¹ s⁻¹, k_d 1.21×10⁻⁴±0.707×10⁻⁴ s⁻¹, K_D 6.47×10⁻¹¹±3.30×10⁻¹¹ M; Q223R: k_a 1.75×10⁶±0.0245×10⁶ M⁻¹ s⁻¹, k_d 1.47×10⁻⁴±0.0505×10⁻⁴ s⁻¹, K_D 8.43×10⁻¹¹±0.407×10⁻¹¹ M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.