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。     

槟榔碱对3T3-L1脂肪细胞脂代谢的影响

目的：观察槟榔碱对3T3-L1脂肪细胞脂代谢的影响并探讨其可能机制。方法：采用经典的"鸡尾酒"法诱导3T3-L1前脂肪细胞分化成熟,随后用不同浓度的槟榔碱（0、25、50、100 μmol/L）处理成熟脂肪细胞72 h。72 h后,四甲基偶氮唑盐（MTT）法检测细胞的活性;油红O染色观察胞浆内脂滴情况;Western blot检测脂肪酸合成酶（FAS）、甘油三酯脂肪酶（ATGL）、激素敏感性脂肪酶（HSL）蛋白表达。结果：诱导分化成熟的脂肪细胞胞浆内可见大量脂滴;MTT显示：0~100 μmol/L槟榔碱对脂肪细胞活力无显著影响;油红O染色后脂质含量测定结果表明槟榔碱能减少成熟脂肪细胞中脂质含量;Western blot结果显示：与0 μmol/L组（对照组）相比,槟榔碱可显著降低脂肪细胞内FAS的蛋白表达,增加ATGL和HSL的蛋白表达;其中以50 μmol/L组最为显著。结论：槟榔碱使脂肪细胞脂解增强,可能与降低脂质合成关键酶FAS的表达,增加脂质分解代谢关键酶ATGL和HSL的表达有关。     

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.     

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。     

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。     

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。     

NF-κB p65在高糖诱导3T3-L1脂肪细胞胰岛素抵抗中的作用

建立高糖诱导胰岛素抵抗的细胞模型,研究高糖对3T3-L1脂肪细胞NF-κB p65表达及转位的影响。诱导成熟的3T3-L1脂肪细胞与5.0mmol/L的葡萄糖含或不含0.6nmol/L的胰岛素(LGIns 组与LGIns-组)或者与25.0mmol/L葡萄糖含或不含0.6nmol/L的胰岛素(HGIns 组与HGIns-组)培养18h,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western印迹检测总NF-κBp65及核NF-κB p65的表达,用激光扫描共聚焦(CLSM)对NF-κB p65进行定位显示。结果显示,仅HGIns 组,即3T3-L1脂肪细胞与25.0mmol/L葡萄糖含0.6nmol/L的胰岛素培养18h后,胰岛素刺激的葡萄糖转运减少55%(P<0.01),同时Western印迹和CLSM均显示NF-κB p65核转位增加(P<0.01),但对3T3-L1脂肪细胞总NF-κB p65的表达无明显影响(P>0.05)。研究结果表明,只有在胰岛素(0.6nmol/L)存在的条件下,高糖(25.0mmol/L)才可以诱导胰岛素抵抗,其分子机制可能与其刺激NF-κB p65的核转位,调节相关基因的表达有关。     

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。     

MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究 *

目的 探究miR-186-5p对小鼠3T3-L1前脂肪细胞增殖,分化的影响及其潜在的分子机制.方法: qRT-PCR检测miR-186-5p在不同周龄小鼠白色脂肪组织及3T3-L1前脂肪细胞增殖分化过程中的表达变化;通过脂质体将miR-186-5p mimics,inhibitors转染入增殖液或分化液培养的3T3-L1细胞后,利用CCK-8,EdU和qRT-PCR检测3T3-L1前脂肪细胞增殖变化,油红O染色观察其脂滴形态;通过生物信息软件TargetScan和双荧光报告系统分别对miR-186-5p靶基因进行预测和确认.结果: (1)miR-186-5p在1~6周龄小鼠的白色脂肪组织及3T3-L1前脂肪细胞自然分化过程中表达量均逐渐上调.(2)与阴性对照相比,mimics或inhibitors转染分别显著地促进或抑制了miR-186-5p的表达.(3)过表达miR-186-5p后,3T3-L1前脂肪细胞的增殖速率减慢,脂滴增大增多;而抑制miR-186-5p后,3T3-L1前脂肪细胞增殖速率增快,脂滴数量减少,且粒径变小.其中过表达miR-186-5p显著地降低了野生型Wnt5a和Mapk1 3'-UTR活性,而突变相应的绑定位点可解除该抑制作用.结论: miR-186-5p可抑制3T3-L1前脂肪细胞增殖,且通过直接靶向Wnt5a和Mapk1以促进其分化为成熟脂肪细胞.     

柽柳黄素对3T3-L1脂肪细胞胰岛素抵抗的影响及AMPK信号通路的作用机制

为探讨柽柳黄素对3T3-L1脂肪细胞胰岛素抵抗的影响及AMPK信号通路的作用机制,本研究利用地塞米松诱导3T3-L1脂肪细胞,建立胰岛素抵抗模型,通过给药后检测细胞对Glu的摄取量和细胞内TG的含量,并采用qRT-PCR对AMPK信号通路中相关基因进行检测,利用分子对接软件对AMPK信号通路中相关蛋白进行分子对接,进一步采用Western blot进行蛋白检测。研究结果表明,当柽柳黄素作用48 h后,高低剂量组均显著增加细胞对Glu的摄取(P<0.01),高剂量组显著降低细胞内TG含量(P<0.05);作用机制显示柽柳黄素具有显著提高AMPK(P<0.01)和降低FAS(P<0.05)基因的表达,能与FAS蛋白具有较好的分子对接,可增加P-AMPK、P-ACC、P-PKB和PPARα和抑制FAS蛋白的表达。该研究说明柽柳黄素可增强胰岛素抵抗模型3T3-L1脂肪细胞对Glu的摄取,降低TG在细胞内的含量,其作用机制可能与AMPK信号通路中相关基因和蛋白调节有关。     

AFM对3T3-L1前脂细胞及其骨架的可视化研究

目的:为脂肪细胞分化提供数据,加深对3T3-L1细胞分化机制的了解.方法:应用AFM对3T3-L1前脂细胞的形貌、超微结构、机械性能和细胞骨架进行了可视化研究.结果:3T3-L1细胞舒展,伪足丰富,膜表面有大小不一的斑块和突起.通过统计分析得出3T3-L1细胞的高低差,均方根粗糙度、平均粗糙度和平均高度分别为622.3nm、77.34nm、55.80nm、393.1nm;AFM针尖与细胞膜表面的相对粘弹力为95.10±19.41pN,平均硬度为2.36±0.39mN/m,杨氏模量为4.85±0.99kPa.AFM对3T3-L1细胞骨架成像,观察到骨架南排列整齐的大纤维束和细小的微纤维以及颗粒状蛋白组成,形成网络结构.结论:细胞形貌结合细胞的机械性能可知3T3-L1细胞生长状态良好,细胞的移动迁移能力强.     

Inhibitory Effects of Fucoidan in 3T3-L1 Adipocyte Differentiation

Fucoidan is a group of sulfated fucose-containing polysaccharides that derived from non-mammalian origin such as marine brown algae, the jelly coat from sea urchin eggs, and the sea cucumber body wall. However, potential biological activities against obesity from fucoidan were not reported in the literature. The objective of this study was to evaluate protective effect of fucoidan in 3T3-L1 adipocyte differentiation. Preadipocyte 3T3-L1 was treated with 100 and 200 μg/ml fucoidan during adipogenesis. Adipogenesis was determined through Oil Red O staining method and the expression of adipogenic genes aP2, ACC, and PPARγ. Adipogenesis of 3T3-L1 treated with 100 and 200 μg/ml fucoidan were significantly inhibited at 32.8% and 39.7% using Oil Red O staining method, respectively (P < 0.05). Treating the 3T3-L1 cells with 100 and 200 μg/ml fucoidan significantly decreased the expression of aP2 gene by 6.2% and 27.2%, respectively, of ACC gene by 22.2% and 38.2%, respectively, and of PPARγ gene by 44.2% and 69.4%, respectively, compared to adipocyte controls (P < 0.05). The results suggest that fucoidan could be used for inhibiting fat accumulation, which is mediated by decreasing aP2, ACC, and PPARγ gene expression.     

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.     

MiR-185 inhibits 3T3-L1 cell differentiation by targeting SREBP-1

Adipogenesis involves a highly orchestrated series of complex events in which microRNAs (miRNAs) may play an essential role. In this study, we found that the miR-185 expression increased gradually during 3T3-L1 cells differentiation. To explore the role of miR-185 in adipogenesis, miRNA agomirs and antagomirs were used to perform miR-185 overexpression and knockdown, respectively. Overexpression of miR-185 dramatically reduced the mRNA expression of the adipogenic markers, PPARγ, FABP4, FAS, and LPL, and the protein level of PPARγ and FAS. MiR-185 overexpression also led to a notable reduction in lipid accumulation. In contrast, miR-185 inhibition promoted differentiation of 3T3-L1 cells. By target gene prediction and luciferase reporter assay, we demonstrated that sterol regulatory element binding protein 1 (SREBP-1) may be the target of miR-185. These results indicate that miR-185 negatively regulates the differentiation of 3T3-L1 cells by targeting SREBP-1, further highlighting the importance of miRNAs in adipogenesis.     

Esculetin induces mitochondria-mediated apoptosis in 3T3-L1 adipocytes

Adipose tissue mass is determined by the volume and the number of adipocytes and is subjected to homeostatic regulation involving cell death mechanisms. We investigated the effects of esculetin, a coumarin compound, on apoptotic signaling in 3T3-L1 adipocytes. Esculetin treatment induced an increase in expression of Bax with a concomitant decrease of Bcl-2 in a time-dependent manner. Esculetin treatment also resulted in translocation of cytochrome c from mitochondria to cytosol and cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP)-1, resulting in the accumulation of an 85 kDa cleavage product in a caspase-dependent manner. Furthermore, esculetin selectively altered the phosphorylation state of members of the MAPK superfamily, causing dephosphorylation of extracellular signal-regulating kinase 1/2 (ERK1/2) and hyperphosphorylation of c-Jun-N-terminal kinase (JNK). In addition, an inhibitor of the JNK MAP kinase pathway, SP600125, reduced esculetin-induced cytochrome c release. These results indicate that esculetin mediated adipocyte apoptosis involves the mitochondrial pathway. Esculetin thus decreases adipocyte number by initiating this apoptotic process in 3T3-L1 adipocytes. This work was supported by the Georgia Research Alliance, AptoTec, Inc., and by the Georgia Research Alliance Eminent Scholar endowment held by CAB.