关岭牛MyoD玉基因启动子报告质粒的构建及活性验证

为了克隆关岭牛MyoD玉基因启动子并验证其启动活性,根据GenBank中牛的MyoD玉基因序列设计PCR引物,用PCR技术扩增牛MyoD玉基因的启动子,构建重组克隆载体pUCmT-MyoD玉;并通过PCR扩增、限制性酶切、测序及生物信息学分析对阳性克隆进行鉴定;构建报告质粒pGL3-MyoD玉,并将其转染小鼠C2C12、3T3-L1细胞系,检测其24 h后的双荧光素酶活性。实验结果获得了关岭牛MyoD玉基因启动子,其序列长度为993 bp,并成功构建了MyoD玉启动子报告质粒;双荧光素酶活性检测表明pGL3-MyoD玉在小鼠C2C12细胞中的表达是pGL3空载体40.65倍,在小鼠3T3-L1细胞中的表达是空载体的1.13倍,MyoD玉启动子在小鼠C2C12细胞中的表达高于3T3-L1细胞(**p<0.01)。结果表明关岭牛MyoD玉基因启动子具有启动活性,在小鼠骨骼肌细胞中特异性表达。     

Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes

Stress hormone is known to play a vital role in lipolysis and adipogenesis in fat cells. The present study was carried out to evaluate the effect of epinephrine on adipogenesis in the 3T3-L1 cells. The investigation on adipogenesis was done in both mono and co-cultured 3T3-L1 cells. 3T3-L1 preadipocytes and C2C12 cells were grown independently on transwell plates and transferred to differentiation medium. Following differentiation, C2C12 cells transferred to 3T3-L1 plate and treated with medium containing 10 μg/ml of epinephrine. Adipogenic markers such as fatty acid binding protein 4, peroxisome proliferator activating receptor, CCAAT/enhancer-binding protein, adiponectin, lipoprotein lipase and fatty acid synthase mRNA expressions were evaluated in the 3T3-L1 cells. Epinephrine treatment reduced adipogenesis, evidenced by reducing adipogenic marker mRNA expression in the 3T3-L1 cells. In addition, glycerol accumulation and oil red-O staining supported the reduced rate of adipogenesis. Taking all together, it is concluded that the stress hormone, epinephrine reduces the rate of adipogenesis in the mono and co-cultured 3T3-L1 cells. In addition, the rate of adipogenesis is much reduced in the co-cultured 3T3-L1 cells compared monocultured 3T3-L1 cells.     

TRIM72, a novel negative feedback regulator of myogenesis, is transcriptionally activated by the synergism of MyoD (or myogenin) and MEF2

TRIM72 is known to be involved in the negative feedback regulation of myogenesis by targeting insulin receptor substrate-1. Here, we found that TRIM72 was more highly expressed in oxidative muscle with the higher activity of MEF2, compared to glycolytic muscle. Indeed, TRIM72 promoter contained an evolutionarily conserved MEF2 site juxtaposed to E-box. TRIM72 promoter activity was decreased by the site-directed mutagenesis of either E-boxes or a MEF2 site and synergistically enhanced by MyoD (or myogenin) and MEF2, which were associated with proximal E-box, and MEF2 site of the TRIM72 promoter, respectively. Taken together all these data, we concluded that the synergism of MyoD (or myogenin) and MEF2 is necessary for TRIM72 expression during C2C12 differentiation.     

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。     

小鼠Myf5真核表达载体的构建及其在细胞中的定位

目的：获得小鼠生肌调节因子Myf5基因并构建pEYFP-C1真核表达载体,观察Myf5在小鼠C3H10T1/2细胞中的定位。方法：利用PCR获得Myf5基因克隆到pEYFP-C1载体中,利用脂质体将构建的表达载体转染C3H10T1/2细胞,荧光观察融合蛋白的表达。结果：从小鼠cDNA文库中得到760bp的myf5的CDS序列后,重组到pEYFP-C1载体中并转染C3H10T1/2细胞,荧光显示Myf5蛋白定位在细胞核中。结论：Myf5载体成功构建并在小鼠C3H10T1/2细胞表达,证明了Myf5蛋白定位于细胞核,为进一步研究Myf5与其他蛋白的相互作用奠定了基础。     

Oxidative stress provokes atherogenic changes in adipokine gene expression in 3T3-L1 adipocytes

Increased oxidative stress has been associated with obesity-related disorders. In this study, we investigated how oxidative stress, in different ways of exposure, regulates gene expression of various adipokines in 3T3-L1 adipocytes. Exposure to 100-500microM H(2)O(2) for 10min, as well as exposure to 5-25mU/ml glucose oxidase for 18h, similarly decreased adiponectin, leptin, and resistin mRNAs, and increased plasminogen activator inhibitor-1 mRNA. Secretion levels of adipokines were also changed by oxidative stress in parallel with mRNA expression levels. Although a peak increase in plasminogen activator inhibitor-1 mRNA was achieved between 4 and 8h after exposure to H(2)O(2) for 10min, significant decreases in adiponectin and resistin mRNA were observed after 16h, while leptin mRNA was decreased earlier. Our results suggest that oxidative stress, even of short duration, has a significant impact on the regulation of various adipokine gene expressions favoring atherosclerosis.     

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。     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

Expression and modulation of TUB by insulin and thyroid hormone in primary rat and murine 3T3-L1 adipocytes

tub encodes a protein of poorly understood function, but one implicated strongly in the control of energy balance and insulin sensitivity. Whilst tub expression is particularly prominent in neurones it is also detectable in extraneuronal tissues. We show here, for the first time, expression of TUB protein in rat adipocytes and the murine adipocyte model 3T3-L1 and demonstrate that insulin induces its tyrosine phosphorylation and association with the insulin receptor. TUB expression is regulated developmentally during adipogenic differentiation of 3T3-L1 cells and in response to cell treatment with thyroid hormone or induction of insulin resistance. TUB was upregulated 5- to 10-fold in adipocytes from obese Zucker rats and 3T3-L1 adipocytes that had been rendered insulin resistant, a response that could be antagonised by rosiglitasone, an insulin-sensitising drug. Our data are consistent with a previously unforeseen role for TUB in insulin signalling and fuel homeostasis in adipocytes.     

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.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma

The malignancy of alveolar rhabdomyosarcoma (ARMS) has been linked to expression of the PAX3-FKHR chimeric gene. To understand the effect of this gene, we used RNAi to knock down its expression (without affecting the expressions of either PAX3 or FKHR) in human ARMS cell lines. Down-regulating PAX3-FKHR caused (a) tumor cells to accumulate in the G1 phase, inhibiting the rate of cellular proliferation, (b) a reduction in the levels of the MET, reducing cell motility stimulated by HGF, and (c) induction of the myogenic differentiation gene, myogenin, and muscle differentiation (morphologic change and the expression of muscle specific proteins, desmin, and myosin heavy chain). These results suggest that PAX3-FKHR in ARMS cells promotes malignant phenotypes such as proliferation, motility, and to suppress differentiation.     

c-Jun N-terminal kinase is involved in the suppression of adiponectin expression by TNF-alpha in 3T3-L1 adipocytes

Adiponectin, one of adipokines that is secreted from adipocytes, plays an important role in the regulation of glucose and lipid metabolism. Paradoxically, serum concentrations of adiponectin are decreased in obese and type 2 diabetic patients, although it is produced in adipose tissue. On the other hand, plasma TNF-alpha levels are increased in such subjects. In the present study, the mechanism by which adiponectin is regulated by TNF-alpha was investigated. The decreased adiponectin mRNA levels by TNF-alpha were partially recovered by treatment with a c-Jun N-terminal kinase (JNK) inhibitor or the PPAR-gamma agonist rosiglitazone in 3T3-L1 adipocytes. Interestingly, however, cotreatment with the JNK inhibitor and rosiglitazone led to a recovery of TNF-alpha-mediated adiponectin suppression to the control level. The JNK inhibitor regulated the expression of adiponectin by the increase of PPAR-gamma DNA binding activity and the recovery of its mRNA expression while rosiglitazone acted via a PPAR-gamma independent pathway which remains to be elucidated. These findings suggest that the JNK signaling pathway, activated by TNF-alpha, is involved in the regulation of adiponectin expression.