黄体酮对3T3-L1脂肪前体细胞成脂分化后细胞中UCP2基因表达的影响

目的:观察体外培养条件下3T3-L1脂肪前体细胞诱导分化成的成熟脂肪细胞中解偶联蛋白2(UCP2)mRNA表达水平及黄体酮对其表达的影响。方法:体外培养3T3-L1脂肪细胞,在诱导3T3-L1脂肪细胞分化成熟后,经不同黄体酮浓度10μm/25μM/50μM/75μM/100μM刺激后,抽提总RNA,用RT-PCR检测UCP2 mRNA的表达。结果:黄体酮会促进成熟脂肪细胞中UCP2 mRNA的表达,(P<0.05)其中25μM浓度刺激下UCP2 mRNA表达量最高。结论:体外培养中,黄体酮对成熟脂肪细胞中UCP2 mRNA的表达与调控具有一定的影响。     

下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响

目的:下调脂肪特异性蛋白27(Fsp27)基因表达联合杨梅素干预,观察对3T3-L1细胞中脂质代谢的影响,并探究脂滴发生、发展变化的调控机制。方法:常规培养3T3-L1前脂肪细胞,采用"鸡尾酒"法诱导其分化为成熟脂肪细胞。脂质体法转染sh-Fsp27干扰载体,以杨梅素浓度为100μmol/L的完全培养基干预成熟脂肪细胞72h。油红O染色,观察脂滴形态及大小的变化;酶法测定细胞内甘油及甘油三酯的含量,观察细胞脂质代谢的变化。Western blot检测Fsp27、激素敏感性甘油三酯脂肪酶(HSL)、甘油三酯脂肪酶(ATGL)以及丝裂原活化蛋白激酶(MAPK)信号通路蛋白的表达。结果:1. 3T3-L1细胞诱导分化后,形态由纤维样变成圆形,并伴随有细胞体积的增大。2.与对照组相比,杨梅素组和转染组细胞中甘油三酯含量下降,甘油含量升高(P 0. 05)。与其他三组相比,联合干预组细胞中甘油三酯含量减少,甘油含量增加(P 0. 05)。3.与对照组相比,其余三组细胞内Fsp27蛋白的表达量均降低,ATGL和PPARγ的表达量升高(P 0. 05)。另外,联合干预组和杨梅素组细胞内HSL的表达量和p-p38MAPK/p38MAPK的比值均大于sh-Fsp27组和对照组(P 0. 05)。结论:1. Fsp27基因沉默与杨梅素联合干预可以更大程度地促进脂肪分解代谢。2.杨梅素可通过激活MAPK信号通路,上调HSL和ATGL的蛋白表达来发挥其促脂解的作用; sh-Fsp27干扰载体通过调节PPARγ和Fsp27蛋白的表达,增加ATGL含量来加速脂肪分解。     

绿原酸在小鼠3T3-L1前脂肪细胞分化进程中的作用

目的：探讨绿原酸（CGA）对小鼠3T3-L1前脂肪细胞分化的影响。方法：培养小鼠3T3-L1前脂肪细胞,分别设置空白对照组（CG）、阳性对照组罗格列酮组（RG）、阴性对照组GW9662组 （GG）和绿原酸组（CGA）。油红O染色观察小鼠3T3-L1前脂肪细胞分化后的细胞形态变化以及脂滴形成情况;组织细胞甘油三酯（TAG）酶法测定各组分化后的细胞TAG积累量;实时荧光定量PCR技术（qPCR）检测小鼠3T3-L1前脂肪细胞在分化过程中关键基因PPARγ2 mRNA表达水平。结果：CGA组被油红O染色的区域大于CG组和GG组,但CGA组颜色没有RG组鲜艳,且脂滴形状也与RG组存在差异。CGA组TAG积累量低于CG组和RG组,与CG组比较无显著性差异（P>0.05）,但与RG组比较有显著性差异（P<0.05）。在分化过程中,CGA组和RG组PPARγ2 mRNA的表达量高于CG组和GG组,有显著性差异（P<0.01）,GG组PPARγ2 mRNA的表达量自细胞分化第4d起低于CG组,有显著性差异（P<0.01）。结论：CGA可以促进小鼠3T3-L1前脂肪细胞的分化,同时降低分化后成熟脂肪细胞中TAG的积累量,其机制与分化相关因子PPARγ2的表达有关。     

3T3-L1前体脂肪细胞分化过程中G蛋白偶联受体48的表达研究

目的 观察G蛋白偶联受体48（GPR48）、过氧化物酶体增殖体激活受体g2（PPARγ2）和CCAAT增强子结合蛋白α（C/EBPα）基因在小鼠胚胎成纤维细胞（3T3-L1）前体脂肪细胞诱导分化过程中不同时段表达水平的变化,探讨GPR48在脂肪细胞分化过程的作用。方法 体外培养3T3-L1前体脂肪细胞诱导分化为成熟脂肪细胞,在分化不同时段（第0~14天）,采用Real-timePCR技术检测脂肪细胞中GPR48、PPARγ2和C/EBPα基因信使核糖核酸（mRNA）的表达水平。结果 GPR48基因在3T3-L1前体脂肪细胞诱导分化第2天和第3天表达显著上调,差异均有统计学意义（t=4.12,P=0.015;t=6.21,P=0.003）,分化第6~14天与分化前表达无差异。PPARγ2表达在诱导分化后明显上调,分化第6天达高峰,第10~14天持续处于较高水平并趋于稳定,与诱导前期相比各时段间表达水平差异均有统计学意义（t在4.17~22.65间,P均〈0.01）。C/EBPα表达在诱导分化后明显上调,分化后第3天达高峰,第6~10天持续保持在较高水平,与诱导前期相比各时段表达水平差异均有统计学意义（t在4.38~13.87间,P均〈0.01）,第14天趋于下调,与分化前比较无差异。GPR48基因表达高峰早于PPARγ2和C/EBPα。结论 在3T3-L1脂肪细胞分化过程中PPARγ2和C/EBPα表达变化与脂肪细胞分化、脂质积聚过程相一致。GPR48基因表达高峰早于PPARγ2和C/EBPα,可能参与了脂肪细胞分化的早期过程。     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、没有长开放阅读框架但往往具有mRNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lncRNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pcDNA3.1-RAP3,利用脂质体将pcDNA3.1-RAP3和人工合成的lnc-RAP3的siRNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、qRT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P<0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P<0.05)或极显著(P<0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P<0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P<0.05)或极显著(P<0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

下调Perilipin 1基因表达对3T3-L1细胞脂解的影响

目的:研究下调围脂滴蛋白基因(PLIN1)表达对3T3-L1细胞脂解的影响。方法:采用RNA干扰技术,构建3组阳性及1组阴性sh-PLIN1重组载体,并进行菌液PCR和DNA测序鉴定。Western blot测定PLIN1A蛋白表达,评价载体下调效果。细胞转染有效载体2天后,Bodipy 493/503染色脂滴;酶学方法测定细胞中甘油三酯和甘油含量;Western blot检测甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)及其磷酸化蛋白(p-HSL)的表达。酶联免疫吸附法(ELISA)测定细胞中环磷酸腺苷(c AMP)和蛋白激酶A(PKA)的浓度。结果:各sh-PLIN1干扰载体构建成功,且3组阳性载体均能显著下调PLIN1A蛋白的表达(P0.05)。转染有效载体后,与阴性转染组相比,sh-PLIN1转染组细胞中脂滴减小,甘油三酯含量降低,甘油含量升高,ATGL和HSL相对表达量显著升高(P0.05),p-HSL相对表达量及c AMP、PKA的浓度无显著性差异(P0.05)。结论:下调PLIN1基因表达可加快3T3-L1细胞脂解速率,其可能通过上调ATGL和HSL的表达而实现,c AMP/PKA信号通路对其无明显调节作用。     

Rheb过表达在前体脂肪细胞株3T3-L1分化中的作用

目的:利用前体脂肪细胞株3T3-L1细胞观察mTOR(mammalian target of rapamycin)信号通路中上游调控因子Rheb(Ras homolog enriched in brain)对其分化的影响。方法:利用高表达Rheb的基因重组质粒转染前体脂肪细胞株,3T3-L1。通过蛋白质免疫印迹实验鉴定质粒成功转染细胞后,诱导该细胞脂肪分化。予以分化第8天的3T3-L1细胞油红染色,并检测细胞内甘油三酯的含量。另外,我们用Western blot方法检测脂肪细胞特异性转录因子PPAR-γ(Peroxisome proliferator-activated receptor-γ)和C/EBP-α(CCAAT-enhancer-binding protein-α)的表达情况来研究Rheb在脂肪细胞分化过程中的作用。结果:我们成功构建了高表达Rheb的3T3-L1细胞株,发现高表达Rheb后可以促进脂滴的生成,油红O染色有显著区别,与对照组相比Rheb高表达组的三酰甘油含量明显升高(P0.05);C/EBP-α和PPAR-γ等脂肪细胞特异性的转录因子蛋白表达量与对照组相比也均有升高(P0.05)。结论:Rheb基因作为mTOR通路上游调控因子,可以促进脂肪细胞的分化。     

MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

辣椒碱对3T3-L1 前脂肪细胞葡萄糖摄取的影响

目的:探讨辣椒碱对3T3-L1前脂肪细胞葡萄糖摄取的影响。方法:不同浓度的辣椒碱作用于3T3-L1前脂肪细胞,采用MTT测定细胞活性,GLU Test试剂盒法测定葡萄糖摄取,Western Blot法检测葡萄糖转运蛋白1(GLUT-1)表达的变化。结果:25μM辣椒碱作用72 h和50μM、100μM辣椒碱作用48 h、72 h,可显著抑制3T3-L1细胞增殖,6.25、12.5、25μM辣椒碱作用可显著促进3T3-L1细胞的葡萄糖摄入,Western Blot结果显示辣椒碱能够显著增加GLUT1蛋白表达量,差异均具有统计学意义(P0.05)。结论:低剂量辣椒碱具有降糖作用,其作用机制可能与增加GLUT-1蛋白表达有关。     

Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

构建脂肪特异性蛋白27(Fat-specific protein of 27,Fsp27)基因沉默载体,研究沉默Fsp27基因表达对3T3-L1细胞脂解的影响,并对其作用机制进行探究。采用RNAi技术,构建Fsp27基因真核干扰载体,下调Fsp27基因的表达。“鸡尾酒”法诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞。脂质体转染脂肪细胞,油红O染色脂滴,酶法测定细胞中甘油及甘油三酯的含量。Western blot法检测细胞中Fsp27、HSL、ATGL和PPARγ的蛋白表达。Western blot结果显示:阳性sh-Fsp27干扰载体均能有效下调Fsp27的表达,且伴随细胞内ATGL和PPARγ的表达量升高(P<0.05),其中sh-Fsp27-2的沉默效果最好;酶学方法检测结果显示:阳性sh-Fsp27干扰组细胞中甘油三酯含量下降,甘油含量升高(P<0.05);油红O染色结果发现:空白对照组与阴性对照组均有大脂滴堆积,阳性sh-Fsp27组小脂滴分布广泛,未见明显的大脂滴。sh-Fsp27-2组基因沉默载体的沉默效果最好,Fsp27基因沉默可以加快3T3-L1细胞的脂解速率,其主要是通过抑制脂滴融合和增强ATGL酶的水解来完成对脂解的调控。     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

Studies on the cell treatment conditions to elicit lipolytic responses from 3T3-L1 adipocytes to TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin

Wasting syndrome is one of the hallmark symptoms of poisoning by TCDD (=dioxin), which is associated with the massive loss of adipose tissue and serum hyperlipidemia in vivo. Yet, the most widely used in vitro cell model 3T3-L1 adipocyte has not been useful for studying such an action of TCDD because of the difficulty of inducing their mature adipocytes to respond to TCDD to go through lipolysis. Here, we made efforts to find the right cell culture and treatment conditions to induce mature 3T3-L1 adipocytes to go through lipolysis, which is defined as events leading to reduction of lipids in adipocytes. The optimum condition was found to require 7-day differentiated adipocytes being subjected to DMEM medium containing TCDD (but without insulin) for 5 day incubation with two medium changes (the same composition) on incubation days 2 and 4. After 24 h, the early effect of TCDD on adipocytes was predominantly on inflammation, particularly induction of COX-2 and KC (IL-8), which is accompanied by upregulation of C/EBPbeta and delta. The sign of TCDD-induced lipolysis starts slowly and by incubation day 3, a few markers showed modestly significant changes. By day 5 of incubation, however, many markers show highly significant signs of lipolytic changes. Although this process could take place without exogenous macrophages or their cytokines, addition of exogenous TNFalpha considerably synergized this action of TCDD. In conclusion, under a right condition, 3T3-L1 adipocytes were found to respond to TCDD to go through lipolysis. The early trigger of such a response appears to be activation of COX-2, which is amplified by TNFalpha.     

Efficient adenovirus transduction of 3T3-L1 adipocytes stably expressing coxsackie-adenovirus receptor

3T3-L1 adipocytes have proven difficult to transfect with plasmid-encoded cDNAs or even infect with virally-derived cDNAs. We have developed and characterized a 3T3-L1 adipocyte cell line stably expressing the truncated receptor for coxsackievirus and adenovirus receptor (CAR) for its ability to be infected with adenoviruses at a low multiplicity of infection (m.o.i.). Using green fluorescent protein driven by the cytomegalovirus promoter in adenovirus fiber type 5 we compared infection efficiencies of CAR adipocytes versus the parental 3T3-L1 adipocytes. As assessed by immunofluorescence, CAR adipocytes were infected at approximately 100-fold greater efficiency than regular 3T3-L1 adipocytes. The efficiency of transduction for the CAR adipocytes was >90% at multiplicities of infection of 50 whereas standard adipocytes were poorly transduced even at an m.o.i. of 2000. Since many investigators studying insulin action use 3T3-L1 adipocytes, we compared CAR adipocytes versus regular adipocytes and showed that the two cell lines were similar with respect to insulin stimulation of insulin receptor, MAPK, and Akt phosphorylation and basal- and insulin-stimulated glucose transport. In addition, CAR adipocytes accumulated GLUT4 and SCD1 proteins during the adipogenesis program with the same time course as regular 3T3-L1 adipocytes. Lastly, CAR adipocytes produced and secreted the adipose-specific hormone Acrp30. These data suggest 3T3-L1CARDelta1 adipocytes are virtually indistinguishable from their parental cells, but demonstrate a significant advantage with improved efficiency of adenoviral transduction for gain or deletion of function studies.     

Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes

Objective : Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic. Research Methods and Procedures : Mature 3T3‐L1 adipocytes were incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were quantified using an MTS‐based cell viability assay and an enzyme‐linked immunosorbent assay for single‐stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen‐activated protein kinases extracellular signal‐regulating kinase 1/2 (ERK) and c‐Jun‐N‐terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase‐3, translocation of apoptosis‐inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116‐kDa poly(ADP‐ribose) polymerase (PARP)‐1. Results : Ajoene induced apoptosis of 3T3‐L1 adipocytes in a dose‐ and time‐dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116‐kDa PARP‐1 in a caspase‐independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N‐acetyl‐l ‐cysteine effectively blocked ajoene‐mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP‐1. Discussion : These results indicate that ajoene‐induced apoptosis in 3T3‐L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen‐activated protein kinases, degradation of PARP‐1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。