地塞米松和胰岛素调节猪脂肪细胞SOCS-3、OB、GLUT4和PPARγ基因的表达

猪是研究糖尿病最理想的模型动物,研究胰岛素和胰岛素抵抗是研究糖尿病的重要环节。为明确SOCS-3在胰岛素抵抗中的作用,分别用100nmol／L的胰岛素,300nmol／L的地基米松处理原代培养的猪脂肪细胞诱导胰岛素抵抗;利用半定量RT-PCR技术分别检测SOCS-3、OB、GLUT4和PPARγ基因表达变化。结果发现,胰岛素增加了GLUT4、SOCS-3和PPARγ基因的表达,对OB基因表达变化没有显著性影响;地塞米松诱导的胰岛素抵抗状态下OB和SOCS-3基因表达水平升高,而GLUT4和PPARγ基因表达水平显著下调。研究结果表明,GLUT4基因表达量水平的升高可能是由于PPARγ的高表达引起,SOCS-3基因的不同表达水平对胰岛素信号的抑制效果不同。地塞米松诱导的胰岛素抵抗不仅表现在对葡萄糖转运的抑制,也反映在抑制了胰岛素信号;而SOCS-3基因可能是消除胰岛素抵抗的一个有效靶基因。     

干扰视黄醇结合蛋白4对猪脂肪细胞PI3K/Akt信号通路的影响

视黄醇结合蛋白4(Retinol binding protein 4,RBP4)是一种脂肪细胞分泌因子,其表达水平的升高与胰岛素抵抗及Ⅱ型糖尿病等疾病密切相关,但具体作用机制尚不清楚。为明确此机制,通过包装RBP4干扰慢病毒并侵染猪前体脂肪细胞。运用胰岛素激活及诱导胰岛素抵抗模型,利用QRT-PCR及Western blotting方法检测RBP4的干扰效率及处理组PI3K/Akt信号通路相关基因的表达。结果显示RBP4的基因及蛋白的干扰效率达到60%(P<0.01)以上。进一步研究发现在胰岛素诱导及胰岛素抵抗的情况下,LH1-shRBP4干扰后可显著提高胰岛素信号通路AKT2、PI3K、GLUT4和IRS1基因mRNA的表达;明显促进AKT2、PI3K和IRS1蛋白的磷酸化;提高AKT2、PI3K和GLUT4基因的总蛋白水平。总之,RBP4干扰通过上调PI3K/Akt胰岛素信号通路相关因子的表达及其磷酸化水平,提高了胰岛素敏感性。此研究将为胰岛素抵抗相关疾病的治疗提供新思路。     

灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

慢病毒载体介导的脂肪细胞SOCS-3RNA干扰及对瘦素反应的研究

体内存在瘦素抵抗是大多数肥胖者的共同特征,但如何缓解瘦素抵抗目前还无有效方法.通过应用慢病毒载体介导的RNA干扰技术对大鼠脂肪细胞中瘦素信号转导通路的负反馈抑制因子—细胞因子信号转导抑制因子-3(suppressor of cytokine signaling 3,SOCS-3)基因进行敲减,观察其对大鼠重组瘦素作用的反应.研究发现,阴性对照慢病毒和SOCS-3shRNA慢病毒对成熟脂肪细胞的感染率约为80%.利用real-time PCR和Westernblot法检测发现SOCS-3 shRNA慢病毒通过在成熟脂肪细胞内表达shRNA而造成SOCS-3基因在mRNA和蛋白水平的敲减,有效率分别为72%和57%;经50nmol/L瘦素处理6h后,感染阴性对照病毒的脂肪细胞SOCS-3mRNA表达有明显升高(P0.05),而感染SOCS-3 shRNA慢病毒的脂肪细胞SOCS-3mRNA表达则无明显变化(P0.05).结果表明,利用SOCS-3shRNA慢病毒去敲减脂肪细胞中SOCS-3的表达可能对于解除肥胖者外周瘦素抵抗具有一定的作用.     

SOCS-3过表达对红系基因表达的影响

目的:建立能稳定、高效表达细胞因子信号转导抑制因子-3(SOCS-3)的细胞株SOCS-3-K562,为探讨SOCS-3在造血发育中的作用奠定基础。方法:通过重组慢病毒系统感染人红白血病细胞K562,采用流式细胞分选术,根据绿色荧光蛋白表达情况,获得稳定高表达SOCS-3的K562细胞;利用实时荧光定量PCR和蛋白质印迹实验,比较分选获得的细胞与对照细胞的SOCS-3表达差异;利用半定量PCR检测SOCS-3表达升高对K562红系发育相关基因GATA-1、β-globin表达水平的影响。结果:构建了人SOCS-3慢病毒表达载体;与对照组相比,通过流式细胞分选获得的K562细胞的SOCS-3基因表达水平升高8.05倍,蛋白表达水平升高3倍;SOCS-3表达升高后,K562细胞的GATA-1、β-globin基因表达受到明显抑制。结论:SOCS-3在造血发育中有重要的调控作用,而对其表达进行改变将在规模化的造血细胞定向诱导研究中发挥重要作用。     

慢性高剂量胰岛素刺激猪脂肪细胞脂肪分解

为研究慢性高剂量胰岛素对猪脂肪细胞脂肪分解的影响及其分子机制, 分化的猪脂肪细胞在PKA(Protein kinase A)或ERK(Extracellular signal-related kinase)抑制剂预处理或不处理的情况下, 再用不同浓度的胰岛素(0、200、400、800、1600 nmol/L)处理不同时间(24、48、72、96 h), 通过测定甘油释放量检测脂肪细胞的脂解率; 采用RT-PCR和Western blotting检测perilipin A和PPARg2的mRNA和蛋白表达。结果显示, 慢性高剂量胰岛素以剂量和时间依赖性的方式刺激猪脂肪细胞的脂肪分解, 并削弱脂肪细胞对异丙肾上腺素刺激的脂解应答; 同时显著下调perilipin A和PPARg2的mRNA及蛋白表达; 另外, PKA和ERK抑制剂均显著抑制胰岛素刺激的脂肪分解, 但仅ERK抑制剂显著逆转perilipin A基因表达的下调。由此推测, 慢性高剂量胰岛素通过ERK通路抑制perilipin A的表达, 进而刺激猪脂肪细胞的脂肪分解。     

中国仓鼠自发性2型糖尿病基础代谢特征及相关基因的表达差异

目的 探究自发性2型糖尿病中国仓鼠糖脂代谢、体成分、昼夜运动及新陈代谢等基础代谢特征和相关基因在骨骼肌、肝中的表达情况。方法 根据中国仓鼠空腹血糖(FBG)和餐后血糖(PBG)值,选取对照组(FBG≤4.5 mmol/L且PBG<6.0 mmol/L)与糖尿病组(FBG≥6.0 mmol/L且PBG>7.0 mmol/L),测定动物体重、血糖、血脂、血清胰岛素含量及糖耐量,分析动物体成分,昼夜运动及新陈代谢特征,检测相关基因葡萄糖转运蛋白4(glucose transporter 4,Glut4)和过氧化物酶体增殖激活受体-γ(peroxisomeproliferative activated receptor-γ,Pparg)在骨骼肌和肝中的表达情况。结果 与对照组相比,中国仓鼠糖尿病组血糖、血脂含量增加,血清胰岛素含量和胰岛素抵抗指数(homeostasis model assessment of insulin resistance,HOMA-IR)增大,体脂率降低,摄食量和白天活动量增加,热量消耗增大。PPARG在肝和骨骼肌中的mRNA和蛋白表达水平显著增加;GLUT4在骨骼肌中的mRNA和蛋白表达水平显著降低。结论 自发性2型糖尿病中国仓鼠属于糖脂代谢异常,能产生胰岛素抵抗的非肥胖型2型糖尿病动物模型,GLUT4的下调可能与骨骼肌中异常的糖代谢及胰岛素抵抗有关,而上调的PPARG可能有利于机体胰岛素抵抗状态的缓解。     

抑制Hedgehog信号通路促进C3H10T1/2间充质干细胞成脂分化

激活Hedgehog信号通路可抑制间充质干细胞成脂分化,但抑制Hedgehog信号通路是否可促进脂肪细胞分化研究结果却并不一致.本研究采用环靶明诱导C3H10T1/2细胞成脂分化,并以国际公认的成脂诱导剂混合物（胰岛素、地塞米松、吲哚美辛和IBMX）诱导细胞分化作为参考. qRT-PCR结果显示,在10 μmol/L环靶明(cyclopamine)处理的C3H10T1/2细胞中,Hedgehog信号通路各基因相对表达量显著下降,而成脂分化调控基因PPARγ,C/EBPα和成脂分化标志基因FABP4相对表达量显著升高（P < 0.05). 与此一致,Western印迹结果表明,在环靶明处理的C3H10T1/2细胞中,Hedgehog信号通路中的Shh蛋白和Gli1蛋白表达水平显著下降,成脂分化相关的PPARγ、C/EBPα和FABP4蛋白表达水平显著升高（P < 0.05）. 此外,油红O染色方法证明,环靶明处理可诱导C3H10T1/2细胞成脂分化.以上研究结果提示,抑制Hedgehog信号通路对小鼠胚胎间充质干细胞的成脂分化具有促进作用,并可能为瘦肉型猪的培育和猪肉品质调控研究提供参考依据.     

猪脂肪前体细胞分化过程中聚脂相关基因的表达模式

本实验采用胶原酶消化法分离猪皮下脂肪前体细胞,用含850 nmol/L 胰岛素和50 nmol/L地塞米松的诱导培养液进行诱导,采用油红O提取法测定了细胞中的甘油三酯含量,同时采用实时定量RT-PCR方法检测了细胞分化过程中聚脂相关基因的表达.结果显示:转录因子PPAR γ和C/EBP β在诱导后12 h即迅速表达,SREBP-1 mRNA表达水平在诱导后12 h出现显著下调,随后逐渐升高,96 h达到最高水平;脂肪合成相关酶基因GPDH、FAS、ACC和LPL呈现出与SREBP-1相似的表达模式;脂肪酸转运相关基因aP2、FAT、FATP1与VLDLR的表达量随着细胞分化过程的延长而不断增加,并且与细胞内甘油三酯的含量变化高度相关.本实验结果表明,PPAR γ、C/EBP β和SREBP-1可能是调控猪脂肪前体细胞分化的关键转录因子.猪皮下脂肪组织在聚脂过程中,在分化早期可能以脂肪细胞自身合成脂肪酸为主,而后期则主要依赖细胞外脂肪酸的跨膜转运.这些结果可能有助于揭示脂肪细胞的分化调控规律.     

胰岛素和瘦素抵抗的重要调控因子:细胞因子信号转导抑制因子-3

细胞因子信号转导抑制因子-3(Suppressor of Cytokine Signaling-3,SOCS-3)是细胞信号转导生理抑制剂家族重要成员之一,主要参与负调控生长激素、白介素(IL),肿瘤坏死因子(TNF)等细胞因子信号转导.最近发现,SOCS-3在胰岛素和瘦素抵抗中具有重要调控功能,且与糖尿病关系密切,是未来糖尿病基因治疗的新靶点.该文综述了近年来SOCS-3结构及其对胰岛素和瘦素抵抗的调控机制研究进展,并探讨SOCS-3在糖尿病中的作用.     

Definitions,measurements, and classifications of stimuli,situations, and environments

A review is presented of issues relevant to the definition, measurement, and classification of stimuli, situations, and environments. Problems such as the lack of adequate definitions of concepts, error and bias in measurement procedures, confusion between measurement of a concept and measurement of its behavioral effects, and the lack of agreement among alternative measures are emphasized. It is suggested that concepts be defined in terms of objective characteristics while allowing for the study of the transactional relationship between organism and environment. The work of the ethologists in defining stimuli while studying their relationship to different organismic states and situational contexts is emphasized in this regard. Following Brunswik, it is also suggested that wherever possible there be a representative sampling of variables in natural settings. Note from the editors: From time to time, Human Ecology will publish a review article. Our first in this series is a review by a psychologist of basic definitional and conceptual problems in environmental studies.This paper was prepared while the author was a Visiting Research Fellow at the Educational Testing Service. The support of ETS and my colleagues in the Division of Psychological Studies is gratefully acknowledged. The review was also supported in part by a grant from the Rutgers University Research Council.     

Exercise, dobutamine, and combined atropine, norepinephrine, and epinephrine compared

We compared the cardiovascular effects evoked in conscious dogs by 1) submaximal exercise; 2) infusion of dobutamine (40 micrograms X kg-1 X min-1); and 3) infusion of a combination of atropine (0.15 mg/kg), norepinephrine (0.19 micrograms X kg-1 X min-1), and epinephrine (0.05 micrograms X kg-1 X min-1). Myocardial O2 demand, as estimated by the double product (heart rate X systolic blood pressure), was similar during all three interventions. Cardiac output and heart rate increased significantly (P less than 0.05) during each of the three interventions. Arteriovenous O2 difference and total body O2 consumption, however, increased only during submaximal exercise. Although myocardial blood flow increased similarly during each of the three interventions, blood flow to skeletal muscle and the tongue increased only during exercise. Exercise and the combined infusion of atropine, norepinephrine, and epinephrine produced similar increases in blood flow to the diaphragm and similar decreases in blood flow to the stomach. These changes in blood flow were associated with appropriate changes in vascular resistance. Additionally, blood flow to the brain, kidney, adrenal glands, liver, and intestine did not change during any of the three interventions. Thus, in dogs, submaximal exercise, infusion of dobutamine, and infusion of a combination of atropine, norepinephrine, and epinephrine to evoke a given level of estimated myocardial O2 consumption produce similar increases in cardiac output, heart rate, and myocardial blood flow. In contrast, the changes in total body O2 consumption, arteriovenous O2 difference, regional blood flow, and regional vascular resistance that occur during each of these three interventions are different.     

Indians, Markets, and Rainforests: Theory, Methods, and Analysis

Indians, Markets, and Rainforests: Theory, Methods, and Analysis. Ricardo A. Godoy. New York: Columbia University Press, 2001. 274 pp.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

Incorporating Science,Technology, Fairness,and Accountability in Environmental,Health, and Safety Decisions

The Precautionary Principle is in sharp political focus today because (1) the nature of scientific uncertainty is changing and (2) there is increasing pressure to base governmental action on more “rational” schemes, such as cost-benefit analysis and quantitative risk assessment, the former being an embodiment of ‘rational choice theory’ promoted by the Chicago school of law and economics. The Precautionary Principle has been criticized as being both too vague and too arbitrary to form a basis for rational decision making. The assumption underlying this criticism is that any scheme not based on cost-benefit analysis and risk assessment is both irrational and without secure foundation in either science or economics. This paper contests that view and makes explicit the rational tenets of the Precautionary Principle within an analytical framework as rigorous as uncertainties permit, and one that mirrors democratic values embodied in regulatory, compensatory, and common law. Unlike other formulations that reject risk assessment, this paper argues that risk assessment can be used within the formalism of tradeoff analysis—a more appropriate alternative to traditional cost-benefit analysis and one that satisfies the need for well-grounded public policy decision making. This paper will argue that the precautionary approach is the most appropriate basis for policy, even when large uncertainties do not exist, especially where the fairness of the distributions of costs and benefits of hazardous activities and products are a concern. Furthermore, it will offer an approach to making decisions within an analytic framework, based on equity and justice, to replace the economic paradigm of utilitarian cost-benefit analysis.