JAZF1基因过表达对鼠肝细胞糖脂代谢相关基因表达的影响

目的探讨JAZF1过表达对鼠肝细胞IAR-20及Hepa1-6糖脂代谢相关基因的影响。方法克隆鼠JAZF1编码区并构建pIRES2-EGFP-JAZF1表达载体,利用脂质体法将目的基因转染到鼠肝细胞IAR-20及Hepa1-6中,采用实时荧光定量PCR测定各处理组JAZF1 mRNA表达情况,以及糖脂代谢相关基因SREBP1、AOC、FAS、HSL、PPARa、ATGL、G1uT-1、G1uT-4和细胞因子FGF-21mRNA的变化情况。结果JAZFl基因在鼠肝细胞中过表达导致脂代谢基因SREBP1,ACC,FAS表达降低（P〈0．01）,PPARa和HSL表达增加（P〈0．01）,对AXG-L无影响（P〉0．05）,使糖转运相关基因GluT-1表达增加（P〈0．01）,GluT-4无显著变化（P〉0．05）,对FGF-21无影响（P〉0．05）。结论JAZF1过表达可以抑制肝细胞的脂肪生成,促进脂肪分解,并可能增加基础葡萄糖转运。     

未知功能基因JAZF1过表达对3T3-L1脂肪细胞糖脂代谢的影响

目的 观察JAZF1基因(Juxtaposed with another zincfinger gene 1)过表达对3T3-L1脂肪细胞糖脂代谢相关基因的影响.方法 采用实时荧光定量PCR(RT-QPCR)法检测JAZF1 mRNA在健康C57BL/6J小鼠多种组织表达分布情况;构建JAZF1真核表达载体并瞬时转染3T3-L1细胞,RT-QPCR法检测JAZF1、糖脂代谢相关基因mRNA的表达;用Western印迹法测定各组细胞JAZF1蛋白水平;油红O染色比色法检测细胞内脂质积聚的变化.结果 转染48 h后,JAZF1转染组脂肪细胞中,JAZF1 mRNA及蛋白表达明显高于阴性对照和空载组,激素敏感脂肪酶(HSL)mRNA表达水平明显增加(P<0.05),脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、类固醇调节元件结合蛋白1(SREBP1)mRNA相对表达量明显降低(均P<0.01),脂肪细胞甘油三酯酶(ATGL)、葡萄糖转运子1(GLUT1)、GLUT4 mRNA表达并无明显改变;油红O染色显示JAZF1转染组细胞内脂质积聚明显降低(P<0.05).结论 JAZFl在C57BL/6J小鼠多种组织均有表达,提示其可能在维持正常生理功能中起着一定作用.3T3-L1细胞过表达JAZF1可减少脂质合成、增加脂解,并可明显改善脂质积聚,可能是肥胖和糖尿病治疗的一个潜在靶点.     

JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢的影响

目的 探讨JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢相关基因的影响.方法 构建JAZF1小发夹RNA (shRNA)表达载体并转染3T3-L1细胞,实时荧光定量PCR(RT-QPCR)和蛋白印迹法检测JAZF1 mRNA和蛋白水平的表达;氢三放射示踪法检测3T3-L1细胞糖摄取率;蛋白印记法检测糖、脂代谢相关基因蛋白水平;油红O染色检测脂肪细胞甘油三酯(TG)含量变化.结果 成功构建JAZF1-shRNA;转染脂肪细胞48 h后,JAZF1 mRNA和蛋白水平明显低于对照组(P＜0.05);氢3放射性示踪法显示转染组葡萄糖摄取率明显降低(P＜0.05);PPAR-γ蛋白表达升高(P＜0.05),激素敏感脂肪酶(HSL)、内脏脂肪素(Visfatin)、胰岛素诱导基囚-2 (Insig-2)蛋白表达降低(均P＜0.05);油红O染色显示JAZF1转染组细胞内脂质积聚明显,比对照组升高约25％(P＜0.05).结论 JAZF1基因抑制可减少基础糖转运,增加脂质与胆固醇合成,减少脂质分解并减少相关脂肪细胞因子的表达.     

Exendin-4对脂肪细胞分化及糖脂代谢相关基因mRNA表达的影响

目的探讨Exendin-4对3T3-L1前脂肪细胞的分化及糖脂代谢相关基因mRNA表达的影响。方法体外培养3T3-L1前脂肪细胞,在脂肪细胞分化成熟过程中的不同时期分别用Exendin-4等干预,采用油红O染色,观察脂肪细胞分化及脂质积聚情况;采用荧光定量PCR检测脂肪细胞糖脂代谢标志基因GLUT-4、PPARγ、HSLmRNA表达水平。酶法测定脂肪细胞的甘油三酯含量。结果分化成熟的脂肪细胞经油红O染色可见细胞质内大片脂滴呈亮红色,而未分化细胞不被油红O染色。在脂肪细胞分化第0天和第6天用Exendin-4干预,脂肪细胞内TG的含量较空白组增加（P〈0．01）,GLUT-4、HSL、PPARγ mRNA的表达上调（P〈0．01）;在脂肪细胞分化的第12天干预,Exendin-4对肪细胞分化及相关基因mRNA的表达与空白组无明显差异。结论Exendin-4促进脂肪细胞的分化并上调糖脂代谢相关基因GLUT-4、PPARγ、HSL mRNA表达,可能为Exendin-4抗糖尿病的部分作用机制。     

抵抗素结合多肽对3T3-L1脂肪细胞分化、脂代谢及葡萄糖转运体4基因表达的影响

目的观察抵抗素结合多肽（RBP）对3T3-L1脂肪细胞分化、脂代谢及葡萄糖转运体4（GLUT-4）基因表达的影响。方法构建大鼠抵抗素真核表达载体并转染3T3-L1前体脂肪细胞，获得稳定表达抵抗素基因细胞株；采用台盼蓝排斥试验，确定理想的RBP干预浓度，于诱导细胞分化第0天加入培养液；采用油红O染色，观察脂肪细胞分化及脂质积聚情况；采用RT-PCR技术检测脂肪细胞分化标志基因及GluT-4基因表达变化；采用全自动生化仪比色法，检测脂肪细胞内TG和游离脂肪酸FFAs含量的变化。结果（1）RBP浓度10^-12mol／L时，脂肪细胞活细胞数比例较高，且细胞形态无明显改变。（2）RBP对正常脂肪细胞分化进程无明显影响，RBP虽未影响抵抗素稳定表达脂肪细胞内脂滴的出现时间，但细胞内脂滴的数目明显减少。（3）RBP对正常脂肪细胞分化标志基因及抵抗素稳定表达细胞分化早期标志基因Pref-1的表达无明显影响，但明显下调抵抗素稳定表达细胞分化中晚期标志基因C/EBPα和FAS的表达水平。（4）RBP对正常脂肪细胞内TG、FFAs含量无影响，但可显著降低抵抗素稳定表达脂肪细胞内的TG、FFAs含量。（5）RBP干预对正常脂肪细胞及抵抗素稳定表达脂肪细胞中GluT-4基因的表达水平均无显著影响。结论RBP对正常3T3-L1脂肪细胞的分化、脂代谢、GluT-4基因表达均无明显影响，但能有效拮抗抵抗素基因，显著促进3T3-L1脂肪细胞分化及脂代谢。     

肺癌肿瘤抑制因子1基因过表达对肝细胞糖脂代谢相关基因表达的影响

探讨肺癌肿瘤抑制因子1(TSLC1)基因表达上调对小鼠肝癌细胞Hepal-6糖脂代谢相关基因mRNA表达的影响.结果 显示,TSLC1基因过表达降低脂代谢基因脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)的表达(P<0.05或P<0.01),增加脂肪组织甘油三酯水解酶(ATGL)的表达(P<0.05),对激素敏感脂肪酶(HSL)、葡萄糖转运蛋白(GLUT)1和GLUT4的表达无显著影响,提示TSLC1过表达可能抑制肝细胞的脂肪生成,促进脂肪分解.

Abstract：

The effects of tumor suppressor in lung cancer-1(TSLC1)upregulation on gene expressions involved in glucose and lipid metabolism in Hepa1-6 cells were investigated.The results showed that TSLC1 overexpression decreased fatty acid synthase and acetyl CoA carboxylase expressions(P<0.05 or P<0.01),increased adipose triglyceride lipase expression(P<0.05),and did not change hormone-sensitive lipase,glucose transporter (GLUT)1,and GLUT4 expressions.These results suggest that TSLC1 overexpression may promote lipolysis and inhibit adipogenesis in liver cells.     

ATF-4过表达对HepG2细胞脂质从头合成的影响

目的观察内质网应激相关因子激活转录因子(ATF)4过表达对肝细胞脂质从头合成通路的影响。方法转染针对HepG细胞ATF-4 siRNA的过表达质粒,分为未转染组、阴性对照组、ATF-4上调组(ATF-4~+组),测定各组HepG2细胞内三酰甘油水平,油红O染色观察细胞脂质沉积,检测各组肝脏脂质从头合成上游转录因子固醇调节元素结合蛋白(SREBP)-1c及下游脂质合成的关键酶乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FAS)、硬酯酰辅酶A去饱和酶(SCD)-1的基因水平,并检测ACC、FAS、SCD-1的蛋白表达水平。结果 ATF-4~+组ATF-4的表达水平比未转染组、阴性对照组显著升高(P0.01);ATF-4+组较阴性对照组TG升高40%(P0.01),油红O染色示脂滴明显增多。ATF-4~+组SREBP-1c基因表达显著增加(P0.05),ACC、FAS、SCD-1的基因表达显著增加(均P0.01),ACC、FAS、SCD-1的蛋白表达增加(均P0.05)。结论 ATF-4过表达可引起HepG2细胞三酰甘油沉积,并引起脂质从头合成通路激活,提示ATF4通过刺激脂质从头合成通路而在肝脏脂质沉积中发挥作用。     

葡萄糖和胰岛素对3T3-L1脂肪细胞内脏脂肪素mRNA表达的影响

目的研究不同浓度葡萄糖和胰岛素对3T3-L1脂肪细胞中内脏脂肪素（Visfatin）mRNA表达的影响。方法通过real—time RT-PCR方法检测不同浓度葡萄糖和胰岛素培养下3T3-L1脂肪细胞Visfatin mRNA的表达。结果葡萄糖增加了3T3-L1脂肪细胞Visfatin mRNA的表达;胰岛素降低其表达。结论葡萄糖和胰岛素对3T3-L1脂肪细胞中Visfatin mRNA的表达有凋控作用。     

性激素对3T3-L1脂肪细胞Visfatin表达的影响

目的 观察雌二醇、睾酮和孕酮对3T3-L1脂肪细胞Visfatin mRNA和蛋白表达的影响.方法 10-8mol/L～ 10-6 mol/L 雌二醇、睾酮或孕酮作用于3T3-L1成熟脂肪细胞和前脂肪细胞,孵育过夜后收集细胞,分别采用RT-PCR和Westem blot检测Visfatin mRNA和蛋白的表达情况.结果 在3T3-L1成熟脂肪细胞,与对照组相比,雌二醇和睾酮分别使Visfatin mRNA表达增加24％(1.74±0.31比1.40 ±0.18,P＜0.05)和28％(1.65±0.90比1.29±0.69,P＜0.05);而孕酮不影响成熟脂肪细胞Visfatin mRNA表达.雌二醇轻度增加成熟脂肪细胞Visfatin蛋白表达,但无统计学差异;10-6 mol/L睾酮使成熟脂肪细胞Visfatin蛋白表达增加134％(0.61±0.40比0.26±0.05,P＜0.05).与雌二醇和睾酮不同,孕酮使成熟脂肪细胞Visfatin蛋白表达下调32％(0.19±0.02比0.28±0.02,P＜0.05).在前脂肪细胞,与对照组相比,10-7 mol/L和10-6mol/L雌二醇使Visfatin mRNA表达增加70％(1.04±0.38比0.61±0.16,P＜0.01)和123％(1.36±0.41比0.61±0.16,P＜0.01);睾酮使Visfatin mRNA表达增加76％(1.02±0.24比0.58±0.36,P＜0.05);孕酮使前脂肪细胞Visfatin mRNA表达增加2.6倍(1.53±1.01比0.42 ±0.14,P＜0.05).结论 性激素通过促进或抑制脂肪细胞Visfatin基因或蛋白的表达,参与调节上述激素引起的脂肪细胞胰岛素抵抗的病理生理过程.     

小干扰RNA沉默脂肪酸合酶基因对人肝细胞株HepG2脂代谢的影响

目的:研究基因干扰技术沉默脂肪酸合酶(FAS)基因后对人肝细胞株HepG2细胞内外脂质含量及脂代谢相关基因表达的影响。方法:合成3对针对FAS mRNA不同位点序列的小干扰RNA(siRNA),分别转染至人肝细胞株HepG 2,实时荧光定量聚合酶链反应(PCR)检测空白对照组(HepG2细胞不经过任何处理)、阴性对照组(转染没有任何基因干扰作用的阴性siRNA)、FAS-siR NA-1转染组、FAS-siRNA-2转染组和FAS-siRNA-3转染组的FAS mRNA的表达。蛋白免疫印迹(Western blot)法检测蛋白质的表达水平,筛选出沉默效果最佳的一对siR NA。将最有效siRNA转染至HepG2细胞,48 h后测定细胞内外甘油三酯及总胆固醇含量以及脂代谢相关基因的mRNA表达水平。结果:25 nmol/L si RNA和3μl/孔(24孔板)转染试剂的转染效率最高。FAS-siRNA-3对HepG2的FAS基因的沉默效果最好,FAS-si RNA-3转染组的FAS在mRNA水平和蛋白水平分别降低为空白对照组的(52.33±3.07)%和(51.57±3.14)%。FAS-siRNA-3沉默FAS基因后HepG2的细胞内和细胞外培养液中甘油三酯含量显著低于空白对照组,细胞外总胆固醇含量显著低于空白对照组。与空白对照组比较,FAS-siRNA-3转染组HepG2的肝脂酶基因mRNA表达水平显著升高(P0.0001),微粒体甘油三酯转运蛋白基因mRNA表达水平显著降低(P0.05),差异均有统计学意义。结论:FAS基因沉默可显著降低HepG2细胞内甘油三酯、细胞外培养液甘油三酯和总胆固醇的含量,需进一步的体内外实验加以验证。     

Effects of 1,25-dihydroxyvitamin D3 on osteoblastic MC3T3-E1 cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was examined for a possible stimulative effect on osteoblastic MC3T3-E1 cells. During the early period of culture, 1,25-(OH)2D3 had a stimulative effect. During the growth phase, however, the steroid had little effect on either the protein or DNA content of the cultures. 1,25-(OH)2D3 increased bone-liver-kidney-type alkaline phosphatase activity in a dose-related manner up to a concentration of 5 pg/ml; the increase was 2.2-fold over the control value. Studies on the effect of actinomycin D or cycloheximide treatment indicated that the vitamin may enhance de novo synthesis of ALP. The steroid also stimulated type I collagen production dose dependently via an increase in collagen synthesis rather than by inhibition of collagen degradation. MC3T3-E1 cells have a specific receptor for 1,25-(OH)2D3 which has a dissociation constant of 4.17 X 10(-11) M and a sedimentation coefficient of 3.67S. The receptor concentration varied with the period of culture, being higher during the growth phase and lower at confluence, but its affinity did not change. The results indicate that 1,25-(OH)2D3 has a direct specific anabolic effect on osteoblastic cells in vitro during the growth phase and that this effect is related to receptor concentration.     

Antiplatelet agent cilostazol potentiates adipocyte differentiation of 3T3-L1 cells

Cilostazol is an antiplatelet drug, which has beneficial effects in treatment of intermittent claudication and decreases serum triacyiglycerol level in these patients. In this study, we examined adipogenic potency of cilostazol using 3T3-L1 preadipocyte cell line because cilostazol is one of the tissue specific phosphodiesterase (PDE) inhibitors. Addition of cilostazol into the differentiation medium including insulin and dexamethasone, induced the adipocyte differentiation without isobutyl methylxanthine (IBMX). Compared with the cells incubated with vehicle, the cells treated with cilostazol contain much more lipid droplets in the cells 6 days after induction of differentiation. Adipocyte specific gene like stearoyl-CoA desaturase was strongly induced after addition of cilostazol. C/EBPbeta, which is induced by IBMX was also induced by cilostazol. These findings suggest a possibility that adipogenic effect of cilostazol is one of the mechanisms, by which this agent decreases blood triacylglycerol level in the intermittent claudication patients.     

Lipoprotein lipase suppression in 3T3-L1 cells by an endotoxin-induced mediator from exudate cells.

Conditioned medium from cultures of mouse peritoneal exudate cells incubated wih endotoxin contains a mediator that markedly suppresses (greater than 90%) lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) activity in differentiating 3T3-L1 mouse preadipocytes. The effect is dependent upon the amount of mediator and is evident as early as 30 min after the addition of the mediator-containing medium to 3T3-L1 cell cultures. Neither endotoxin nor conditioned medium from cultures of exudate cells not exposed to endotoxin shows the presence of the mediator. Lysates of the exudate cells are also unable to suppress the lipase activity. Increasing the amount of insulin does not reverse this suppression, even at 1000 times the concentration used for standard experiments. The lipoprotein lipase suppression mediator present in the conditioned medium of endotoxin-treated exudate cells is heat labile and has an apparent molecular weight of at least 12,000. The mediator does not inhibit lipoprotein lipase activity directly nor does it affect the half-life of enzyme activity released in the medium. The present study demonstrates that endotoxin promotes the release of a mediator from exudate cells that suppresses the activity of lipoprotein lipase in 3T3-L1 preadipocytes.     

Lipoprotein lipase suppression in 3T3-L1 cells by a haematoprotozoan-induced mediator from peritoneal exudate cells

Lysates of the haematoprotozoa Trypanosoma brucei or Plasmodium berghei stimulated murine peritoneal exudate cells to release a mediator, which suppressed lipoprotein lipase activity in differentiating 3T3-L1 preadipocytes. The parasite-induced mediator suppressed the activity of cell surface lipoprotein lipase up to 39% in a dose dependent manner. By impairing the activity of cell surface lipoprotein lipase, this mediator acts to inhibit the uptake of fatty acid, and ultimately the accumulation of lipid by the adipocyte. In vivo this defect in triglyceride removal may explain the hypertriglyceridemia commonly observed in haematoprotozoan infections. We suggest that the lipoprotein lipase suppression mediator is produced as a consequence of the immune response to these parasitic protozoa.     

柯萨奇B3病毒对HL-1心肌细胞的影响

目的探讨柯萨奇B3病毒对HL-1心肌细胞影响。方法实时荧光聚合酶链反应检测柯萨奇B3病毒感染的HL-1心肌细胞Bcl-2及Bax的表达：Annexin V-PE凋亡检测试剂盒检测感染的HL-1心肌细胞发生凋亡的比率,并与未感染心肌细胞进行对比。结果感染后4h组（0．199±0．005vs0．104±0．014,P=0．0008）及24h组（0．154±0．005vs0．1044-0．014．P=0．0176）的Bel－2／Bax表达均显著高于未感染组,差异有统计学意义。感染后4h组的Bel-2／Bax表达高于感染后24h组,差异有统计学意义（0．199±0．005孤0．154±0．005,P=0．0009）。感染后4h组（9．160％±0．161％vs．0．908％±0．015％,P〈0．0001）、12h组（6．305％±0．231％vs．0．908％±0．015％．P〈0．0001）、24h组（3．260％±0．162％vs0．908％±0．015％,P〈0．0001）的心肌细胞凋亡率显著高于未感染组,差异均有统计学意义。感染后4h组的心肌细胞凋亡率高于感染后12h组（9．160％±0．161％vs．6．305％±0．231％．P〈0．0001）和24h组（9．160％±0．161％vs．3．260％±0．162％,P〈0．0001）,差异均有统计学意义。结论柯萨奇B3病毒导致HL-1心肌细胞的凋亡,在感染4h后增加尤为明显。     

Insulin-stimulated methylaminoisobutyric acid uptake in 3T3-L1 fibroblasts and fat cells

3T3-L1 fibroblasts and fat cells have been extensively used to study the development of insulin-stimulated glucose and lipid metabolism during adipocyte differentiation in vitro. In this paper we explore the ability of insulin to stimulate amino acid uptake in 3T3-L1 cells using the nonmetabolizable amino acid analog methylaminoisobutyric acid (MAIB). In differentiated 3T3-L1 fat cells, a 12-h preincubation with insulin was required for maximal stimulation of MAIB uptake. In contrast, in the undifferentiated fibroblasts, insulin stimulation peaked between 6 and 8 h and then declined significantly. Maximal stimulation of MAIB uptake in the differentiated fat cell exceeded that in the fibroblast phenotype. This increased ability of insulin to stimulate MAIB uptake in fat cells appeared within the first day after removal of the differentiation medium. 3T3-L1 fat cells were 30 times more sensitive to the effects of insulin on MAIB transport than the undifferentiated fibroblasts. These findings are consistent with previous data on insulin-stimulated deoxyglucose uptake, in that the increased sensitivity to insulin with differentiation is more than can be accounted for by the increase in receptor number. The activity of porcine proinsulin indicates that this stimulation reflects the known characteristics of the insulin receptor. The stimulation of MAIB uptake by insulin in 3T3-L1 fat cells was blocked by inhibitors of protein synthesis (cycloheximide and puromycin) and mRNA synthesis (actinomycin D). Colchicine, an inhibitor of microtubule function, showed little inhibition of insulin-stimulated MAIB uptake. Insulin stimulation of MAIB uptake was greater when 3T3-L1 cells were preincubated with insulin in the absence of essential amino acids. Basal transport in 3T3-L1 cells was not influenced by the presence or absence of amino acids. Thus, amino acid deprivation appears specifically to enhance the ability of insulin to stimulate amino acid transport in cultured adipocytes.     

Mid-G1 marker protein(s) in 3T3 mouse fibroblast cells.

Quiescent 3T3 mouse fibroblast cells in a state of growth arrest due to serum deprivation were exposed to [14C]isoleucine. The cell cultures were then stimulated by the addition of 10% fetal calf serum. At various times after stimulation, the 14C-labeled cells were exposed to [3H]isoleucine. Cytoplasmic extracts from the double-labeled cells were subjected to sodium dodecyl sulfate/slab gel electrophoresis. By these procedures it was found that the relative rate of synthesis of a protein species of Mr 50,000 increased after stimulation of quiescent cells, reached a maximum at 5 hr, and then decreased before the 3T3 cells began to enter the S phase. The characteristic peaking profile of mid-G1 protein synthesis exhibited by the Mr 50,000 polypeptide can serve as a useful marker for the progression of events in G1 prior to exit into S.     

Knockdown of macrophage migration inhibitory factor disrupts adipogenesis in 3T3-L1 cells

Obesity is a condition in which adipose tissue mass is expanded. Increases in both adipocyte size and number contribute to enlargement of adipose tissue. The increase in cell number is thought to be caused by proliferation and differentiation of preadipocytes. Macrophage migration inhibitory factor (MIF) is expressed in adipocytes, and intracellular MIF content is increased during adipogenesis. Therefore, we hypothesized that MIF is associated with adipocyte biology during adipogenesis and focused on the influence of MIF on adipogenesis. To examine the effects of MIF on adipocytes, MIF expression in 3T3-L1 preadipocytes was inhibited by RNA interference, and cell differentiation was induced by standard procedures. The triglyceride content of MIF small interfering RNA (siRNA)-transfected 3T3-L1 cells was smaller than that of nonspecific siRNA-transfected cells. In addition, MIF knockdown apparently abrogated increases in adiponectin mRNA levels during differentiation. Gene expression of peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT/enhancer binding protein (C/EBP)alpha, and C/EBPdelta decreased with MIF siRNA transfection, but C/EBPbeta expression increased. Cell number and incorporation of 5-bromo-2-deoxyuridine into cells decreased from 1-3 d and from 14-20 h, respectively, after induction of differentiation in MIF siRNA-transfected cells, thus suggesting that MIF siRNA inhibits mitotic clonal expansion. Taken together, these results indicated that MIF regulates differentiation of 3T3-L1 preadipocytes, at least partially, through inhibition of mitotic clonal expansion and/or C/EBPdelta expression.