【作者】 毋文静；

【导师】 刘艳芬；

【作者基本信息】 广东海洋大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 目的本研究旨在探讨抵抗素基因(RETN)在猪前脂肪细胞分化过程中表达水平的变化规律以及胰岛素、葡萄糖、地塞米松对RETN表达水平的影响。方法1、从1日龄仔猪皮下脂肪组织分离前脂肪细胞,于37℃、5%CO2、饱和湿度下进行原代培养,通过MTT法,油红O染色以及测定细胞内脂蛋白脂酶活性鉴定猪前脂肪细胞。2、用油红O染色法观察前脂肪细胞培养3、5、7、10d后细胞形态的变化,并用实时荧光定量PCR检测前脂肪细胞分化过程中RETN基因表达水平的变化。3、将增殖的猪前脂肪细胞随机分为3组,分别培养于含有胰岛素、葡萄糖和地塞米松的培养基中。培养液中胰岛素终浓度为0,10,100和1000 nmol/L;葡萄糖终浓度为0,5,10和20 mmol/L;地塞米松的终浓度为0,10,100和1000 nmol/L。每个浓度设三个重复。接种1d后将培养基更换为测试培养基即加入影响因子的培养基(记为0d),此后每隔2d换液一次。并于换液后的第3、5、10d后取样。通过油红O染色法观察胰岛素、葡萄糖、地塞米松对前脂肪细胞分化的影响,并用实时荧光定量PCR检测胰岛素、葡萄糖、地塞米松对RETN基因表达水平的影响。结果1、前脂肪细胞培养至第3d时,细胞内有少量脂滴出现,此后细胞内脂滴聚集,脂滴数量增加,体积逐渐增大,最后汇集成大脂滴充满细胞。低剂量的葡萄糖(5mM)和地塞米松(10nM)对前脂肪细胞分化的影响不明显,胰岛素、萄糖和地塞米松都有诱导前脂肪细胞分化的作用,随着影响因子的浓度增大而细胞分化速度加快,随培养时间的增加而分化程度增大。2、抵抗素在3,5,7,10d的相对表达量分别是14.31±1.29,5.58±0.29,2.25±0.08,1.61±0.01,相邻两时间点比较抵抗素的表达差异均有显著性意义(P<0.01)。3、与对照组相比,胰岛素能显著促进RETN的表达,此作用具有剂量依赖性;葡萄糖均能提高RETN的表达,且呈时间依赖性。4、地塞米松在低浓度(10nM)的情况下,短时间(3d)内可抑制抵抗素的表达,5天内对抵抗素表达影响较小,与对照组差异不显著(P>0.05);但长时间内可诱导抵抗素的表达,而地塞米松为100nM和1000nM时,对抵抗素表达的诱导作用呈剂量和时间依赖性(P<0.05)。结论1、在细胞分化过程中,抵抗素的表达量显著下降(p<0.01),提示RETN基因与前脂肪细胞脂肪代谢调节有关,其表达水平下调能促进前脂肪细胞分化。2、胰岛素能显著上调抵抗素的表达且呈剂量依赖性(P<0.05),;葡萄糖能诱导抵抗素的表达并呈时间依赖性(P<0.05);低剂量的地塞米松(10nM)在短时间(3d)内可以抑制抵抗素的表达,其表达量下降了4.3%,高剂量地塞米松均能诱导抵抗素的表达。3、尽管胰岛素、葡萄糖、地塞米松都能不同程度地促进抵抗素基因表达,但均不能改变猪前脂肪细胞分化过程中抵抗素表达水平下降的趋势。更多还原

【Abstract】 ObjectivesThe aim of present study was to investigate the resistin gene (RETN) expression during the differentiation of porcine preadipocyte in vitro and to evaluate the effects of different concentration of insulin, glucose and dexamethasone on RETN of porcine preadipocyte.Methods1.The porcine preadipocytes were isolated from subcutaneous fat tissue of 1-day-old piglet and cultured in 37℃, 5%CO2, saturation humidity and subsequently identified by morphology observation, i.e, MTT or Oil Red O staining and detection of lipoprotein lipase( LPL ) activity.2.The primary preadipocytes were cultured in the DMEM/F12 media for 3, 5, 7 and 10 days respectively, the cell differentiation was evaluated by intracellular lipid droplet stained by red oil O, and the RETN gene expression during the preadipocyte differentiation were determined by real-time fluorescence quantitative RT-PCR simultaneously.3. The proliferated preadipocytes were randomly devided into 3 groups and the cells were respectively treated with different doses of insulin, glucose or dexamethasone in the DMEM/F12 media, and the finally concentration of insulin in the media was 0,10,100,and 1000 nmol/L, glucose concentration was 0,5,10 and 20 mmol/L, concentration of dexamethasone is 0,10,100 and 1000 nmol/L, each concentration with 3 repeats. The preadipocytes were cultured in the DMEM/F12 media with insulin, glucose or dexamethasone suplementation for 3, 7 and 10 days respectively, and the cell differentiation was evaluated by intracellular lipid droplet stained by red oil O, and the RETN gene expression were determined by real-time fluorescence quantitative RT-PCR simultaneously.Results1.A few differentiated adipocytes, in which filled with fatty droplet, were observed at 3 d. The differentiation of porcine peradipocyte could be induced by insulin, glucose and dexamethasone. 2. The RETN expression level of the porcine preadipocytes cultured for 3, 5, 7 and 10 days were respectively 14.31±1.29, 5.58±0.29, 2.25±0.08 and 1.61±0.01, which were significantly decined during the cell differentiation.3. The RETN expression of the preadipocyte was significantly increased by insulin supplementation in the media, which was dose-dependent; the RETN expression level were significantly related to the glucose concentration of the midia, and which was relativly higher as the cultured time of the cell prolonged.4. Except for 10nmol/L dexamethasone teatment, the RETN expression of the porcine preadipocyte were sigificantly promoted by of dexamethasone in a positve manner. But the RETN expression was sligtly decreased by 4.3% in the preadipocyte treated with 10nmol/L dexamethasone.Conclusion1.During the preadipocyte differentiation, the level of RETN expression was significantly decreased(P<0.01), RETN gene may be associated with fat metabolism regulation, and the differentiation of porcine preadipocyte can be promoted by down-regulate RETN gene expression.2. The RETN gene expression of the porcine preadipocyte in vitro could be promoted by insulin in a dose-dependent manner(P<0.05). Furthermore, the RETN gene expression could be significantly elevated by glucosein a time-dependent manner(P<0.05). The RETN expression could be inhibited by 10 nmol/L dexamethasone, wheras 100, 1000 nmol/L dexamethasone could up-regulate RETN expression in a dose-dependent and time-dependent manner(P<0.05).3. Although the RETN expression of the preadipocyte could be up-regulated to some extent by insulin, glucose and dexamethasone, the decreasing trend of the RETN expression of the porcine preadiocytes could not be changed by these factors above.更多还原