TNF-α介导的11-β羟类固醇脱氢酶1表达和活性变化对3T3-L1脂肪细胞胰岛素敏感性的影响

目的：观察肿瘤坏死因子α（TNF-α）介导的11-β羟类固醇脱氢酶1（11-β HSD-1）表达和活性变化对3T3-L1脂肪细胞胰岛素敏感性的影响。方法:以TNF-α以及TNF-α分别与阿司匹林、2’-hydroxyflavanone和RU486联合作用3T3-L1脂肪细胞，然后检测细胞11-β HSD-1 mRNA表达和活性以及胰岛素刺激的葡萄糖摄取能力。 结果:TNF-α增加3T3-L1脂肪细胞11-β HSD-1 mRNA表达和活性，同时降低细胞胰岛素刺激的葡萄糖摄取。阿司匹林减轻了TNF-α对11-β HSD-1 mRNA表达和活性的上调作用，并减轻TNF-α对胰岛素刺激的葡萄糖摄取的抑制作用；11-β HSD-1活性特异抑制剂2’-hydroxyflavanone和皮质醇受体拮抗剂RU486也可减轻TNF-α对胰岛素刺激的葡萄糖摄取的抑制作用。结论:TNF-α可通过提高3T3-L1脂肪细胞11-β HSD-1的表达和活性而降低细胞对胰岛素的敏感性。     

小檗碱激活AMPK抑制3T3-L1脂肪细胞分化

目的 探讨小檗碱对3T3-L1脂肪分化的作用是否与激活腺苷酸活化蛋白激酶（AMPK）有关.方法 在3T3-L脂肪细胞分化全程加入小檗碱,以油红O染色检测3T3-L1脂肪细胞胞浆中脂肪的堆积,实时定量PCR检测过氧化物酶体增殖物激活受体γ2（PPARγ2）、CCAAT增强子结合蛋白α（CEBPα）和AMPK的mRNA表达,以Western印迹法检测AMPK和乙酰辅酶A羧化酶（ACC）的磷酸化水平.结果 小檗碱剂量依赖性地抑制3T3-L1脂肪细胞分化,10 μmol/L小檗碱几乎完全抑制胞浆中脂肪的堆积.5 μmol/L小檗碱在脂肪细胞诱导分化1、3、5、7d后均显著降低CEBPα mRNA表达（P〈0.05或P〈0.01）,诱导分化3、5、7d时显著降低PPARγ2的mRNA表达（P〈0.05或P〈0.01）.AMPK的mRNA水平在分化过程中未受小檗碱的明显影响,而小檗碱明显增加其蛋白磷酸化水平,其下游靶基因ACC磷酸化水平也明显增加.结论 小檗碱抑制3T3-L1脂肪细胞的分化可能与其激活AMPK有关.     

促酰化蛋白诱导的前脂肪细胞分化过程中转录因子表达的时序性研究

目的：观察促酰化蛋白（ASP）诱导3T3-L1前脂肪细胞的分化过程，转录因子PPARγ、C/EBPδ、C/EBPα mRNA表达的强度及时序性。 方法： 以3T3-L1前脂肪细胞为实验对象，用ASP代替经典激素鸡尾酒诱导刺激中的胰岛素，即促酰化蛋白、1-甲基-3-异丁基黄嘌呤和地塞米松（ASP+IBMX+DEX）诱导3T3-L1前脂肪细胞分化，分别在诱导分化1 d、2 d、4 d、6 d、8 d收获细胞，采用RT-PCR法检测ASP诱导3T3-L1前脂肪细胞分化过程中转录因子PPARγ、C/EBPδ、C/EBPα mRNA表达的情况。 结果： PPARγ mRNA在诱导分化1 d时有低水平表达，在诱导分化过程中表达逐步升高，在终末分化阶段仍保持高水平表达。C/EBPδ mRNA在诱导分化1 d时有中等水平表达，在诱导分化2 d时表达水平最高，诱导分化4 d时表达明显减少，在诱导分化6 d和8 d，检测不到C/EBPδ mRNA的表达。C/EBPα mRNA在诱导分化1 d仅有低水平表达，在诱导分化过程中表达逐步升高，在终末分化阶段仍保持高水平表达。IBMX+DEX诱导前脂肪细胞分化过程中，PPARγ、C/EBPδ和C/EBPα mRNA分化早期也有一定升高，但明显低于ASP诱导的转录因子的表达。 结论： ASP对转录因子C/EBPδ、C/EBPα和PPARγ表达的时序性影响，可能是ASP诱导前脂肪细胞分化的重要分子机制之一。     

葛根素对3T3-L1细胞PPAR-γ mRNA表达的影响

大量研究表明,葛根素（puerarin,Pue）可明显改善实验性糖尿病胰岛素抵抗,近年来临床上也有了Pue改善糖尿病患者胰岛素敏感性的报道,但是葛根素对糖尿病胰岛素抵抗的作用及其分子机制研究迄今尚未见详细报道。为进一步明确葛根素对改善糖尿病患者胰岛素敏感性影响,本文采用胰岛素／地塞米松诱导大鼠前脂肪细胞株3T3-L1建立胰岛素抵抗模型,观察葛根素对3T3-L1细胞过氧化物酶体增殖物激活受体（PPAR-γ）mRNA表达的影响。     

肿瘤坏死因子α对3T3-L1细胞葡萄糖摄取和IRS-1信号通路的影响

目的：观察TNF-α作用于3T3-L1细胞对胰岛素介导的葡萄糖摄取和IRS-1相关信号通路的影响。方法：TNF-α分别作用于3T3-L1细胞6小时和24小时后用胰岛素刺激，观察细胞葡萄糖摄取，IRS-1酪氨酸、丝氨酸307磷酸化水平以及PKB磷酸化水平。同时观察那巴霉素对TNF-α作用的影响。结果：TNF-α明显抑制胰岛素刺激的葡萄糖摄取、IRS-1酪氮酸及PKB磷酸化，这些作用可被那巴霉索逆转。TNF-α作用6小时对IRS-1蛋白无影响但导致丝氨酸307磷酸化，作用24小时则降低IRS-1蛋白水平，那巴霉素拮抗TNF-α导致IRS-1减少但对丝氮酸磷酸化无作用。结论：TNF-α导致的脂肪细胞胰岛素抵抗与IRS-1酪氮酸磷酸化水平下降密切相关，Rapamycin可以逆转TNF-α的作用。     

罗格列酮对多囊卵巢综合征患者卵巢黄素化颗粒细胞胰岛素受体底物1和2蛋白表达及酪氨酸磷酸化的影响

目的： 研究罗格列酮对多囊卵巢综合征(PCOS)患者卵巢黄素化颗粒细胞胰岛素受体底物-1(IRS-1)和IRS-2蛋白表达及酪氨酸磷酸化的影响。探讨罗格列酮改善PCOS卵巢局部胰岛素抵抗的作用。方法: 收集行IVF-ET治疗的11例PCOS患者（PCOS组）和15例排卵正常患输卵管性不孕患者（对照组）促排卵后黄素化颗粒细胞行体外培养，分别用不同浓度罗格列酮（0、1、10、100、1 000、10 000 nmol/L）处理细胞48 h，采用RT-PCR、免疫印迹（Western blotting）及免疫沉淀法分别检测卵巢黄素化颗粒细胞IRS-1和IRS-2mRNA的表达、蛋白含量及酪氨酸磷酸化水平。结果: （1）与对照比较，PCOS组黄素化颗粒细胞IRS-1mRNA表达及蛋白含量显著增加（P<0.05），IRS-2mRNA表达及蛋白含量显著降低（P<0.05），IRS-1和IRS-2酪氨酸磷酸化水平均显著降低（P<0.05，P<0.05）；（2）不同浓度罗格列酮作用后，PCOS组黄素化颗粒细胞IRS-1mRNA及蛋白表达显著降低，IRS-2mRNA及蛋白表达显著增加，同时IRS-1和IRS-2酪氨酸磷酸化水平也显著增加，而正常对照组黄素化颗粒细胞IRS表达及酪氨酸磷酸化水平无显著变化。结论: PCOS患者卵巢局部存在胰岛素抵抗，其原因可能与IRS-1和IRS-2蛋白表达及酪氨酸磷酸化异常有关；罗格列酮可以通过调整IRS-1和IRS-2表达失衡，提高IRS-1和IRS-2酪氨酸磷酸化水平，改善PCOS患者卵巢局部胰岛素抵抗。     

罗格列酮上调高脂饮食老年大鼠骨骼肌CPTⅠ和PPARγ的表达

目的 观察高脂饮食对老年大鼠骨骼肌肉碱棕榈酰基转移酶Ⅰ（CPTⅠ）和过氧化体增殖物激活受体γ（PPARγ）的表达变化及罗格列酮的干预效果。方法 大鼠随机分为对照组、高脂组（HF）、罗格列酮干预组（RSG）,每组20只。应用清醒状态下正常葡萄糖高胰岛素钳夹技术的葡萄糖输注率评价胰岛素抵抗,用RT-PCR法和Western blot印迹技术检测骨骼肌CPTⅠ和PPARγ的表达。结果 高脂组骨骼肌甘油三酯（TG）升高而葡萄糖输注率明显下降（P<0.01）,骨骼肌CPTⅠ和PPARγ表达明显降低(CPTⅠb mRNA 3.16±0.59vs1.30±0.18) (PPARγmRNA1.48±0.25vs1.04±0.33)（P<0.05, P<0.01）,罗格列酮干预组显著缓解高脂组上述变化(CPTⅠb mRNA1.30±0.18vs2.84±0.72) (PPARγmRNA1.04±0.33vs2.13±0.42)（P<0.01）。结论 罗格列酮上调高脂饮食老年大鼠骨骼肌CPTⅠ和PPARγ的表达,可能是减少骨骼肌内脂质堆积改善胰岛素抵抗的机制之一。     

神经肽Y促进3T3L1前脂肪细胞的增殖和分化

目的:观察神经肽Y对3T3L1前脂肪细胞增殖和分化的影响,并对其机制进行初步探讨。方法:3T3-L1细胞由3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine,IB-MX)、胰岛素和地塞米松联合诱导分化,2 d后将细胞分为空白对照组(未加任何诱导剂组)、经典诱导组(胰岛素诱导)和NPY干预组(10-8、10-9、10-10mol/L NPY)。培养的第7、12天用相差显微镜观察各组细胞形态的变化,培养第12天用油红O染色观察脂肪细胞分化程度。MTT检测细胞增殖。Western blot检测脂肪细胞分化相关基因过氧化物体增殖剂活化受体-γ(peroxisome proliferator-activated receptor gamma,PPAR-γ)、CAAT/增强子结合蛋白-α(CCAAT/enhancer-bind-ing protein-α,C/EBP-α)蛋白的表达。结果:10-8mol/L NPY能促进3T3-L1细胞的分化。10-9mol/L NPY,10-8mol/L NPY均能促进3T3-L1细胞增殖。10-8mol/L NPY增加C/EBPα、PPARγ的表达。结论:NPY促进3T3L1前脂肪细胞的增殖和分化,其机制可能与上调PPARγ、C/EBPα的表达有关。     

PPARγ激动剂对RSV感染的A549细胞TLR3、TNF-α表达的抑制作用

目的 探讨 A549细胞呼吸道合胞病毒（RSV）感染后Toll样受体3（TLR3）及肿瘤坏死因子-α（TNF-α） mRNA和蛋白表达及PPARγ激动剂15-脱氧前列腺素J2（15d-PGJ2）和罗格列酮的干预作用.方法 建立RSV感染的细胞模型,将传代培养的细胞随机分成6组：15d-PGJ2干预组（A）、罗格列酮干预组（B）、RSV组（C）、PDTC组（D）、GW9662组（E）及对照组（F）.各组在培养12、24和48 h后收获上清液和细胞,实时荧光RT-PCR检测TLR3和TNF-α mRNA表达,Western Blot检测TLR3蛋白表达,ELISA法检测上清中TNF-α蛋白水平.结果 与F组相比,各时间点C组TLR3和TNF-α mRNA及蛋白表达均明显升高（均P＜0.01）;与C组相比,A组、B组和D组TLR3和TNF-α mRNA及蛋白表达均明显降低（均P＜0.01）.15d-PGJ2和罗格列酮对TLR3和TNF-α mRNA和蛋白表达的抑制作用在12 h最明显;同一时间点A组和B组TLR3和TNF-α mRNA和蛋白的表达量均呈剂量依赖性下降.结论 RSV感染后TLR3和TNF-α mRNA及蛋白表达升高;罗格列酮和15d-PGJ2能以剂量依赖性方式抑制TLR3和TNF-α mRNA和蛋白表达.     

miR-202通过抑制PGC1β表达促进3T3-L1前脂肪细胞分化

目的观察miR-202对3T3-L1前脂肪细胞分化的影响及可能的机制。方法通过慢病毒感染构建稳定表达AMO-miR-202和乱序对照miRNA细胞系,随后诱导分化。至分化的第9天,油红O染色观察细胞内脂滴的情况;RT-PCR检测过氧化物酶体激活增殖受体γ2(PPARγ2)和a P2的基因表达;Western blot检测PPARγ2、a P2和miR-202靶基因PPARγ辅助活化因子1β(PGC1β)蛋白表达。结果经293T细胞慢病毒包装AMO-miR-202、乱序对照miRNA,荧光显微镜下可见约80%~90%荧光细胞;将上述2组病毒液分别感染3T3-L1前脂肪细胞后,可见约70%~80%荧光细胞。AMO-miR-202组细胞内脂滴及PPARγ2和a P2的mRNA表达显著低于乱序对照组和对照组(P<0.05)。与乱序对照组和对照组相比,AMO-miR-202组PGC1β蛋白表达显著增加(P<0.05),PPARγ2和a P2蛋白表达显著降低(P<0.01),而乱序对照组和脂肪细胞组上述指标无明显差异。结论 miR-202可能通过抑制PGC1β、提高PPARγ2和a P2的表达促进3T3-L1前脂肪细胞分化。     

3-Hydroxy-3-Methylglutaric Aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglecemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

3-Hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin-7

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α⁺, CD8β^-, IL-4 R⁺ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vγ3⁺. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-γδ mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vγ3⁺ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.     

Assembly of the TCR/CD3 complex: CD3ϵ/δ and CD3ϵ/γ dimers associate indistinctly with both TCRα and TCRβ chains. Evidence for a double TCR heterodimer model

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3ϵ with either CD3γ or CD3δ. These dimers assemble with TCRα and TCRβ chains, and finally, the CD3ζ homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3ϵ/CD3γ dimer interacts exclusively with TCRβ and the CD3ϵ/CD3δ dimer with TCRα to form a complex with a single TCRα/β heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3ϵ/CD3γ and CD3ϵ/CD3δ dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCRα-negative T cell line MOLT-4 in which TCRβ is found separately associated with CD3ϵ/CD3γ and with CD3ϵ/CD3δ dimers. Indirectly, our results support a model of stoichiometry in which two TCRα/β heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3γ and anti-CD3δ antibodies from 1 % NP40 and 1 % Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3ζ homodimer. This suggests that CD3ζ mediates the interaction between both TCRα/β heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCRβ that impairs the association of CD3ζ. In this mutant cell line, unlike a control line with wild-type TCRβ, the CD3γ- and CD3δ-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-α/β heterodimers form two hemicomplexes containing either CD3ϵ/γ or CD3ϵ/δ dimers which become associated via the CD3ζ homodimer.