大黄素对3T3-L1前脂肪细胞诱导分化的作用

目的:观察大黄素(emodin)对3T3-L1前脂肪细胞的诱导分化作用,并探讨其可能机制。方法:采用油红O染色、三酰甘油(TG)含量测定及3-磷酸甘油脱氢酶(GPDH)活性测定等方法检测脂肪细胞的分化程度;用实时定量PCR法检测大黄素对脂肪细胞过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA、CCAAT增强子结合蛋白α(C/EBPα)mRNA和脂肪酸结合蛋白ap2mRNA表达的影响;用四甲基偶氮唑盐(MTT)比色法测定3T3-L1前脂肪细胞的增殖;运用表面等离子体共振技术(SPR)测定大黄素与PPARγ2的亲和力。结果:大黄素呈剂量依赖性促进3T3-L1前脂肪细胞的诱导分化,50μmol/L大黄素可使脂肪细胞内TG含量和GPDH活性分别增加约22%及60%。大黄素可显著升高脂肪细胞的PPARγ2mRNA、C/EBPαmRNA及脂肪酸结合蛋白ap2mRNA表达水平。同时,50μmol/L大黄素可使前脂肪细胞的细胞增殖率下降约17%。SPR检测结果显示,大黄素具与PPARγ2结合活性,提示其可能是PPARγ2的配体。结论:大黄素作为PPARγ2的配体,能促进脂肪细胞分化、抑制前脂肪细胞增殖,而该过程可能是通过上调PPARγ2、C/EBPα的表达来实现的。     

高良姜二苯庚烷化合物对3T3-L1前脂肪细胞分化的影响和机制研究

目的 探讨高良姜二苯庚烷化合物对3T3-L1前脂肪细胞分化的影响和机制。方法 本研究选取2022年9月—2023年2月珠海市人民医院·暨南大学附属珠海医院3T3-L1前脂肪细胞为研究对象,使用MTT法评估高良姜二苯庚烷化合物对3T3-L1细胞增殖的影响。通过油红O染色检测分析高良姜二苯庚烷化合物对3T3-L1细胞分化的影响。应用实时定量聚合酶链锁反应（Polymerase Chain Reaction, PCR）方法检测高良姜二苯庚烷化合物对过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator Activated Receptor γ, PPARγ)和CCAAT/增强子结合蛋白α(CCAAT/Enhancer Binding Proteinα, C/EBPα)基因mRNA表达的调控作用。通过Western blot分析检测高良姜二苯庚烷化合物对PPARγ和C/EBPα蛋白表达的影响。结果 高良姜二苯庚化合物（0、0.05、0.2、0.4 g/L）在24、48 h对前脂肪细胞的生长表现为增殖趋势,呈现一定的量效关系,其中高良姜二苯庚烷化合物浓度为0.4 g/L时3...     

shRNA沉默IRS-1基因对鼠前脂肪细胞分化和PPARγ表达的影响

目的观察胰岛素受体底物-1（IRS-1）基因表达受阻对小鼠3T3-L1前脂肪细胞分化和脂质过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响。方法针对小鼠IRS-1基因开放阅读框上的2个区域合成短发夹核糖核酸[shRNA（M和MH）],以pGenesil-1vector为载体构建带绿色荧光蛋白（GFP）靶标的shRNA质粒。以脂质体LipofectaminTM2000介导转染3T3-L1前脂肪细胞,并设阴性对照（HK）载体转染组。细胞转染后,G418筛选稳定表达的阳性克隆并通过免疫印迹法鉴定。应用0.5mmol/L3-异丁基-1-甲基黄嘌呤（IBMX）、10-6mol/L地塞米松（DEX）和5μg/mL胰岛素（Ins）分别诱导小鼠3T3-L1前脂肪细胞空白对照组、HK载体转染组、M和MH转染组分化,在诱导分化的不同时间点收集细胞。用免疫印迹法检测PPARγ的表达。脂肪细胞内脂滴用油红O染色观察。结果与空白对照组、HK组和转染M组IRS-1shRNA质粒的3T3-L1前脂肪细胞相比,转染MH组IRS-1shRNA质粒的3T3-L1前脂肪细胞IRS-1蛋白表达水平降低70%以上;而MH转染组细胞PPARγ蛋白的表达与前3组3T3-L1前脂肪细胞比较无改变。前3组3T3-L1前脂肪细胞诱导分化为成熟脂肪细胞成功,PPARγ蛋白的表达在脂肪细胞分化成熟过程中逐渐增高。油红O染色显示,随着脂肪细胞的分化成熟,桔红色脂滴逐渐增多;而转染MH组IRS-1shRNA质粒的3T3-L1前脂肪细胞经诱导,油红O染色桔红色脂滴显着减少,直到分化的第8天,才见少许桔红色脂滴,且PPARγ蛋白的表达无变化。结论 IRS-1基因沉默后可抑制3T3-L1前脂肪细胞的分化,并在分化过程中抑制PPARγ的表达,说明IRS-1通过调节PPARγ的表达或与PPARγ共同作用在3T3-L1前脂肪细胞的分化中发挥决定性作用。     

3T3-L1细胞与人骨髓间充质干细胞体外成脂及脂肪细胞功能的比较研究

目的:探讨3T3-L1细胞与人骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外成脂及脂肪细胞功能的差异。方法:用密度梯度离心和贴壁培养的方法分离纯化人骨髓MSC,在倒置显微镜下观察细胞形态,体外向脂肪方向诱导分化,并通过油红O染色和吸光度值(OD值)检测其分化程度;qRT-PCR检测过氧化物酶体增殖物激活受体γ(PPARγ)、脂肪酸结合蛋白(FABP4)、CCAAT增强子结合蛋白(C/EBPα)mRNA表达水平;通过诱导的脂肪细胞与THP-1细胞共培养,加入1μg/ml的阿糖胞苷,在48 h测定其对肿瘤细胞的化疗抵抗作用。结果:通过油红O染色和测定吸光度值发现,3T3-L1细胞组脂质聚集量多于MSC组,且OD值大于MSC组(P0.05);qRTPCR结果显示,3T3-L1来源的脂肪细胞的成脂基因PPARγ、FABP4和C/EBPαmRNA的相对表达量高于人骨髓MSC的脂肪细胞(P0.05);共培养实验结果表明,含脂肪细胞组THP-1细胞存活数较对照组多(P0.05),且3T3-L1细胞组与MSC组之间具有统计学差异(P0.05)。结论:3T3-L1细胞的体外成脂能力比人骨髓MSC强;脂肪细胞具有促使THP-1细胞耐受化疗的作用,且在3T3-L1细胞组的作用大于人骨髓MSC组。     

大黄素对脂肪细胞水通道蛋白-7表达的调节效应与其机制研究

目的观察大黄素对脂肪细胞水通道蛋白-7(AQP7)表达的调节效应并探讨其可能机制。方法成熟的3T3-L1前脂肪细胞分为5组,不同浓度大黄素组(1μM、10μM、50μM),空白对照组(不加药,加入同等体积二甲基亚砜),阳性对照组(吡格列酮10μM)干预24 h。分析大黄素对3T3-L1前脂肪细胞的分化影响,采用MTT法检测12h、24 h、48 h脂肪细胞活力。3T3-L1脂肪细胞诱导分化成熟后构建3T3-L1脂肪细胞胰岛素抵抗模型,分为5组,酶比色法测定甘油及葡萄糖摄取量。用Western Blot分析脂肪细胞中AQP7、过氧化物体增殖剂活化受体γ(PPAR-γ)表达量。结果大黄素组与阳性对照组OD值、3T3-L1脂肪细胞胰岛素抵抗模型甘油及葡萄糖摄取率及脂肪细胞中AQP7与PPAR-γ表达量显著高于空白对照组(P0.05),并且大黄素组呈现剂量依赖性,但大黄素组中浓度与阳性对照组之间无显著性差异(P0.05)。大黄素浓度在1~50μM时,3T3-L1前脂肪细胞活力均≥0.95,且浓度为10μM时,3T3-L1前脂肪细胞活力1。结论大黄素能够促进脂肪细胞分化,增加脂肪细胞同时对甘油及葡萄糖摄取,既能改善胰岛素抵抗又不增加体重,并且存在剂量依赖性。     

ANGPTL4基因在3T3-L1前脂肪细胞诱导分化过程中表达水平的变化

目的观察血管生成素样蛋白-4（Angiopoietin-like protein 4,ANGPTL4）基因在3T3-L1前脂肪细胞诱导分化过程中表达水平的变化,探讨ANGPTL4基因与脂肪细胞分化的关系。方法体外培养3T3-L1前脂肪细胞,在3T3-L1前脂肪细胞分化成熟过程中（0-10 d）,采用油红O染色,观察脂肪细胞分化及脂质积聚情况;采用Real-time PCR、逆转录PCR技术检测脂肪细胞分化标志基因（LEPTIN,ADIPONECTIN）和ANGPTL4 mRNA表达;采用Western blot检测ANGPTL4蛋白表达。结果随着脂肪细胞诱导分化逐步深入,油红O染色显示,细胞中脂滴形成逐渐增多,达到占全视野95%以上;同时瘦素（LEPTIN）和脂联素（ADIPONECTIN）基因在诱导后的第2天开始表达并逐步增多,均提示脂肪细胞分化逐步成熟。ANGPTL4 mRNA和蛋白在前脂肪细胞中低表达,分化第2-6天ANGPTL4基因表达显著上调,分化第8-10天ANGPTL4基因表达保持在较高水平并趋于稳定。结论在3T3-L1前脂肪细胞分化过程中ANGPTL4基因表达上调,ANGPTL4基因可能参与了脂肪细胞分化。     

3T3-L1脂肪细胞分化不同阶段抵抗素表达的变化

目的:抵抗素具有直接对抗胰岛素作用,观察3T3-L1脂肪细胞分化不同阶段过程中抵抗素mRNA和蛋白表达的变化。方法:实验于2006-03/10在郑州大学第一附属医院完成。3T3-L1前脂肪细胞由武汉同济医院儿科馈赠。①将3T3-L1前脂肪细胞接种于培养瓶,加入含体积分数为0.1小牛血清的高糖DMEM培养液,在37℃、体积分数为0.05的CO2条件下培养。待细胞融合2d后,加入体积分数为0.1小牛血清的高糖DMEM培养液培养(含0.5mmol/L3-异丁基-1-甲基黄嘌呤、5mg/L胰岛素、1μmol/L地塞米松),诱导3T3-L1前脂肪细胞分化成熟。②分别于诱导分化的第2,4,6,8天采集细胞,采用反转录-聚合酶链反应检测脂肪细胞抵抗素mRNA的表达,以免疫印迹分析方法检测脂肪细胞抵抗素蛋白的表达。结果:①3T3-L1前脂肪细胞分化的形态学变化:诱导分化前细胞呈梭形,胞浆中无脂滴,表现为成纤维细胞样的前脂肪细胞形态,待完全汇合后处于生长停滞期;诱导分化第2天细胞变圆;第4天时细胞变大,变圆,胞浆有少量脂滴出现;第6天脂滴明显增多;至第8天更多的脂滴积累,形成典型的“戒环”样结构。②抵抗素mRNA在3T3-L1脂肪细胞分化不同阶段表达的变化:3T3-L1脂肪细胞抵抗素mRNA在分化的第2,4,6,8天吸光度值分别为0.16±0.03,0.37±0.07,0.63±0.11和0.96±0.17,相邻两时间点比较抵抗素mRNA的表达差异均有显著性意义(P均<0.01)。③抵抗素蛋白在3T3-L1脂肪细胞分化不同阶段表达的变化:3T3-L1脂肪细胞抵抗素蛋白在分化的第2,4,6,8天吸光度值分别为28942.5±1392.7,59326.7±2954.3,107821.6±4325.1和173656.5±8356.7,相邻两时间点比较抵抗素蛋白的表达差异均有显著性意义(P均<0.01)。结论:抵抗素mRNA及蛋白在脂肪细胞分化成熟过程中表达逐渐升高。     

替米沙坦对体外培养脂肪细胞脂联素表达的影响

背景:脂联素对胰岛素抵抗、代谢综合征有着重要的调节作用,其合成受过氧化物酶体增殖物活化受体γ活性的调节,研究表明部分血管紧张素受体拮抗剂可通过激活过氧化物酶体增殖物活化受体γ影响脂联素代谢,但具体机制尚不清楚.目的:探讨替米沙坦对脂肪细胞脂联素表达和分泌的影响,并与坎地沙坦进行比较.方法:体外培养、诱导分化3T3-L1脂肪细胞,分别用空白培养液、坎地沙坦(10 μmol/L)和替米沙坦(10 μmol/L)干预3T3-L1脂肪细胞,采用RT-PCR法、Western blot法和ELISA法检测脂肪细胞脂联素mRNA和蛋白表达及培养液中脂联素的分泌水平.结果与结论:与空白组和坎地沙坦组相比,替米沙坦组脂肪细胞脂联素的mRNA和蛋白表达水平明显增加(P<0.05或P<0.01),但各组间脂肪细胞培养液中脂联素的分泌水平差异无显著性意义(P>0.05).说明替米沙坦可以明显增加脂肪细胞脂联素的表达,但并不增加脂联素的分泌水平,其促进脂联素表达的作用优于坎地沙坦.     

pEGFP-N1-过氧化物酶体增殖物激活受体γ载体构建及在兔骨髓间充质干细胞中的表达

背景:过氧化物酶体增殖物激活受体γ是细胞分化重要的调控因子,通过调节其他转录因子的表达,参与调节骨髓间充质干细胞的分化.目的:构建pEGFP-N1-过氧化物酶体增殖物激活受体γ真核表达载体,体外转染兔骨髓间充质干细胞,为过氧化物酶体增殖物激活受体γ受体功能和基于过氧化物酶体增殖物激活受体γ受体靶点的药物筛选提供分子研究平台.设计、时间及地点:单一样本观察,于2007-09/2008-07在南华大学附属一医院临床研究所完成.材料:选择雄性2-5月龄新两兰大白兔,用丁分离培养骨髓间充质干细胞.方法:应用密度梯度离心法分离骨髓间充质干细胞,贴壁法不断纯化.从正常小鼠的肝组织中提取总RNA,反转录-聚合酶链反应法获得过氧化物酶体增殖物激活受体γcDNA.经Xho Ⅰ,Apa Ⅰ双酶切,定向克隆到真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-过氧化物酶体增殖物激活受体γ.主要观察指标:经酶切分析和测序鉴定重组质粒中过氧化物酶体增殖物激活受体γ基因的完整性和真实性,脂质体介导下体外转染骨髓间充质干细胞,荧光倒置显微镜下观察瞬时表达及转染效率,提取总RNA和总蛋白,进行反转录-聚合酶链反应和Western blot检测.结果:获得的过氧化物酶体增殖物激活受体γ基因片段经酶切和测序鉴定正确,重组质粒经酶切和测序鉴定证实构建正确,成功转染骨髓间充质干细胞,在其中检测到目的基因及蛋白表达.结论:实验成功构建了pEGFP-N1-过氧化物酶体增殖物激活受体γ重组质粒,同时在转染的骨髓间充质干细胞中获得了过氧化物酶体增殖物激活受体γ的高效表达.     

3-hydroxy-3-methylglutaric aciduria: Deficiency of 3-hydroxy-3-methylglutaryl coenzyme a lyase

A marked deficiency of 3-hydroxy-3-methylglutaryl coenzyme A lyase activity is present in cultured skin fibroblasts from a baby with 3-hydroxy-3-methylglutaric aciduria.     

Pharmacokinetics of 3''-fluoro-3''-deoxythymidine and 3''-deoxy-2'',3''-didehydrothymidine in rats.

Concentrations of 3'-fluoro-3'-deoxythymidine (FDT) and 3'-deoxy-2',3'-didehydrothymidine (D4T) in plasma declined in a biexponential fashion. Total clearance of D4T (1.75 +/- 0.22 liters/h/kg; mean +/- standard deviation) was significantly greater than that of FDT (1.19 +/- 0.19 liters/h/kg) owing to greater renal and nonrenal clearances of the former. Steady-state volumes of distribution of FDT (1.20 +/- 0.12 liters/kg) and D4T (1.07 +/- 0.15 liters/kg) were similar.     

Pharmacokinetics of 3''-fluoro-3''-deoxythymidine and 3''-deoxy-2'',3''-didehydrothymidine in rhesus monkeys.

3'-Fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine are nucleoside analogs which inhibit human and simian immunodeficiency virus in vitro. The pharmacokinetic properties of these compounds in rhesus monkeys after intravenous, oral, and subcutaneous administration of the drug were compared. Half-lives, total clearances, and steady-state volumes of distribution of the two drugs were determined. The half-lives for the drugs by the different routes were between 0.58 and 1.4 h. Oral bioavailability of 3'-deoxy-2',3'-didehydrothymidine was incomplete, with an average of 42% +/- 15% of the dose reaching the systemic circulation. Absorption of 3'-fluoro-3'-deoxythymidine after oral administration was variable, with bioavailability ranging from 21 to 95%. Bioavailability after subcutaneous administration ranged from 59 to 77% for 3'-deoxy-2',3'-didehydrothymidine and from 52 to 59% for 3'-fluoro-3'-deoxythymidine. The ratio of concentrations in cerebrospinal fluid and serum for the drugs was about 0.15 at 1 h after drug administration and was independent of the route of administration, suggesting that a nucleoside carrier-mediated process is involved in the transport of these compounds to the central nervous system. Because of the similar metabolism of nucleoside analogs in monkeys and humans, the potential glucuronide formation was assessed. Whereas the glucuronide of 3'-fluoro-3'-deoxythymidine was readily detected in urine, the amount of 3'-deoxy-2',3'-didehydrothymidine glucuronidated was small or not detectable in one-half of the urine samples. Pharmacokinetic parameters for the two drugs were similar to each other and analogous to those for 3'-azido-3'-deoxythymidine in monkeys, suggesting that the same dose and scheduling of the drug can be used for all three compounds in prophylactic and therapeutic efficacy drug studies in rhesus monkeys.     

Antiviral Activity of 3-Deazaguanine, 3-Deazaguanosine, and 3-Deazaguanylic Acid

3-Deazaguanine (ICN 4221), 3-deazaguanosine (ICN 4793), and 3-deazaguanylic acid (ICN 5412) represent a new class of synthetic guanine analogs having antiviral activity. In vitro, nine ribonucleic acid and seven deoxyribonucleic acid viruses were inhibited, including influenza, parainfluenza, rhino-, vesicular stomatitis, adeno-, herpes-, cytomegalo-, vaccinia, pseudorabies, and myxoma viruses. They were effective orally against influenza types A and B and parainfluenza type 1 (Sendai) virus infections in mice, with a therapeutic index of 16 against the latter two viruses. The course of herpes encephalitis was altered only when the drugs were applied directly into the brain. In addition, these drugs were effective inhibitors of Friend leukemia virus-induced splenomegaly in mice; treatment also produced extensions of life in these animals.     

亚急性甲状腺炎33例临床分析

目的 探讨亚急性甲状腺炎的病因、临床表现、诊断及治疗。方法 对33例患者的病史，临床表现，实验室资料及治疗进行总结分析。结果 患病的女性多见，占78．7％，病前无上怂病史记载：所有病例均有甲状腺肿大并触痛，彩超均有甲状腺肿大伴低回声，所有患者经口服泼尼松治疗效果好。结论 为减少漏诊、误诊，对咽痛伴发热病例，按上感治疗无效且持续时间较长应警惕本病。注意触诊甲状腺有无肿痛并进行必要的辅助检查。肾上腺糖皮质激素是治疗本病唯一确切有效药物。     

3-Methylcrotonylglycine excretion in 3-hydroxy-3-methylglutaric aciduria

1. 3-Methylcrotonylglycine was identified in urine from an infant with 3-hydroxy-3-methylglutaric aciduria. 2. The concentration of 3-methylcrotonylglycine in urine was approximately one sixth of that of the other metabolite of 3-methylcrotonyl-CoA, 3-hydroxyisovaleric acid. 3. The presence of both metabolites in the infant's urine indicates an inhibition of 3-methylcrotonyl-CoA carboxylase activity in tissues of the infant.     

In-plane stimulated emission of polycrystalline CH3NH3PbBr3 perovskite thin films

Hybrid organic–inorganic lead halide perovskites have been investigated extensively within the last decades, for its great potential in efficient solar cells and as an ideal light source. Among the studies on stimulated emission (SE), the emission is either out-of-plane for polycrystalline films or in-plane with randomly aligned single microcrystals and nanowires. In this work, we revealed in-plane propagation of SE from bromine-based perovskite polycrystalline thin films (CH₃NH₃PbBr₃, or MAPbBr₃). The output from in-plane SE is an order higher than the out-of-plane emission. It is proposed that large crystalline flakes in the films lead to the in-plane lasing phenomena. The output coupling can be found at grain boundaries, intergrain gaps, and artificial structures. Simulative results support the experimental phenomenon that large crystalline grains are profitable for in-plane propagation and over 90% photons can be sufficiently outcoupled when the gap is larger than a micron. Considering the fabrication and handling convenience, we propose that the MAPbBr₃ thin films can be easily integrated for in-plane applications as the light source for photonic chips etc.

MAPbBr₃ perovskite thin film contains large crystal flakes, which support the in-plane stimulated emission and its propagation within these polycrystalline films. The emission scatters at the natural or artificial edge of the film.     

3-Hydroxy-3-methylglutaric aciduria: a new assay of 3-hydroxy-3-methylglutaryl-CoA lyase using high performance liquid chromatography

A new assay has been developed for 3-hydroxy-3-methylglutaryl-CoA lyase, the final enzyme in the leucine degradative pathway. The assay was performed by incubating lysates of fibroblasts with [glutaryl-3-¹⁴C](d,l)-3-hydroxy-3-methylglutaryl coenzyme A. The products were analysed by high performance liquid chromatography with continuous liquid scintillation counting. This provided simultaneous identification and quantification of one of the enzymatic products, [3-¹⁴C] acetoacetic acid. The mean 3-hydroxy-3-methylglutaryl-CoA lyase activity in fibroblasts from five controls was 732 ± 81 (SD) pmol/min · mg protein. Using this assay, we have studied skin fibroblasts cultured from a patient with 3-hydroxy-3-methylglutaric aciduria and found 3% of normal 3-hydroxy-3-methylglutaryl-CoA lyase activity. The activities in skin fibroblasts cultured from the parents were 46 and 53% of control activity which is consistent with heterozygocity. Kinetic studies of 3-hydroxy-3-methylglutaryl-CoA lyase in skin fibroblasts cultured from two normal subjects yielded K_m values of 14.4 and 18.8 μmol/l for 3-hydroxy-3-methylglutaryl-CoA.     

Low temperature scintillation performance of a Br-doped CH3NH3PbCl3 single-crystalline perovskite

Time response and light yield are two of the most important features of a scintillation detector, and are mostly determined by the luminescence properties of the scintillator. Here we have investigated the radioluminescence (RL) characteristics of a single-crystalline hybrid lead halide perovskite at both room temperature and low temperature. A dual-channel single photon correlation (DCSPC) system with a vacuum chamber is employed for the measurement. A rise time faster than 100 ps and several times enhancement of the crystal scintillation performances at low temperature have been observed. These behaviors demonstrated that bulk solution-grown single crystals of hybrid lead halide perovskites (MAPbCl₃ and Br-doped MAPbBr_0.08Cl_2.92, where MA = CH₃NH₃) can serve as stable scintillating materials for pulsed gamma detectors. In addition, this work provides a pathway for perovskite application and also attracts attention to investigating low-temperature scintillators.

Time response and light yield are two of the most important features of a scintillation detector, and are mostly determined by the luminescence properties of the scintillator.