【作者】 张琨；

【导师】 张声华；

【作者基本信息】 华中农业大学 ， 食品科学， 2007， 硕士

【摘要】 脂肪细胞的过度肥大和增殖是导致肥胖的主要原因，研究前脂肪细胞的增殖及分化有助于深入了解脂肪组织发育的生理学和病理生理学的机理。RAS是一个具有内分泌特性的肽能的系统，AngⅡ作为RAS主要的效应肽在肥胖症及其相关疾病的控制中发挥重要作用，但作用效果的报道尚存在矛盾的，可见关于AngⅡ对前脂肪细胞分化的影响及其机制仍需要进一步探索和研究。因此本文选用3T3-L1小鼠前脂肪细胞系为体外培养模型，采用血清法、血清饥饿法、油红O染色法、分光光度测定法及逆转录．聚合酶链式反应(RT-PCR)技术研究了AngⅡ对3T3-L1前脂肪细胞增殖及分化的影响及机制。1．3T3-L1前脂肪细胞生长及分化的生物学特性采用台盼蓝染色计数法、鸡尾酒诱导法、油红O染色法和分光光度测定法研究了细胞生长曲线、形态学特性、3T3-L1细胞分化过程中胞内脂质含量及GPDH活性。结果表明：3T3-L1细胞在第2 d进入对数生长期，第4 d达到最高峰，第5 d进入了平台期；分化后第4 d细胞内出现戒环状小脂滴，最后融合成单脂滴；3T3-L1细胞分化过程中，胞内脂质含量随分化时间的增加而增加，且分化第6 d后增加迅速。GPDH活性随分化时间的增加而增大，在分化第4 d后迅速增大，第8 d左右达到高峰并维持在较高水平。2．AngⅡ对3T3-L1前脂肪细胞增殖的影响采用血清法和血清饥饿法两种方法，研究了AngⅡ对3T3-L1前脂肪细胞增殖的影响。结果表明0.01μmol／L、0.1μmol／L、1μmol／L、10μmol／L的AngⅡ对3T3-L1前脂肪细胞的增殖无显著性影响(P＞0.05)，提示AngⅡ对3T3-L1前脂肪细胞的增殖无显著性影响。3．AngⅡ对3T3-L1前脂肪细胞分化的影响采用1μmol／L、100 nmol／L、10 nmol／L、1 nmol／L、0.1 nmol／L 5个AngⅡ浓度梯度，研究了AngⅡ对3T3-L1细胞分化后胞内脂质含量和GPDH活性的影响。结果表明100 nmol／L和10 nmol／L浓度的AngⅡ处理细胞8 d后，胞内脂质的总含量分别比对照组增长了62％和21％，GPDH活性在100 nmol／L浓度的AngⅡ处理8 d后显著地增加，比对照组增长了64％，提示AngⅡ可促进3T3-L1细胞分化。4．AngⅡ对3T3-L1前脂肪细胞分化转录因子PPARγ、C／EBPα、SREBPlc mRNA表达的影响运用RT-PCR技术研究了3T3-L1分化过程中AngⅡ对其转录因子PPARγ、C／EBPα、SREBPlc mRNA表达的影响。结果表明100 nmol／L的AngⅡ处理细胞6 d后，PPARγ、C／EBPα、SREBPlc mRNA的相对表达量比对照组分别增加了48％、50％、43％，该结果与AngⅡ增加3T3-L1分化中脂质含量和GPDH活性的结果相吻合，提示AngⅡ促进3T3-L1细胞分化的分子调控机制可能与上调生脂的转录因子PPARγ、C／EBPα和SREBPlc mRNA的表达有关。更多还原

【Abstract】 The primary reason of obesity is hypertrophy and hyperplasia in adipocyte. Studieson proliferation and differentiation of preadipocyte will reveal the physiological andpathophysiological mechanisms underlying adipose tissue development. RAS is apeptidergic system with endocrine characteristics. As the main effector peptide of theRAS, AngⅡis involves in control of obesity and other correlative disease. However,paradoxical results about the effects of AngⅡwere reported. 3T3-L1 preadipocyte wasused for culture in vitro model in this study. The effect of AngⅡon proliferation anddifferentiation in 3T3-L1 preadipocyte is investigated by serum or serum starvationmethod, Oil Red O staining, spectrophotometry and reverse transcription- polymerasechain reaction （RT-PCR） in this article.1. The biological characteristic of growth and differentiation in 3T3-L1To draw the growth curve though cell counting by trypanblau staining, the resultindicated that growth of cells is extremely vigorous in DMEM containing 10% new borncalf serum, 37℃、5%CO₂, the exponential phase of growth began from the second day andplatform phase was achieved in the fifth day. In microscope, the global lipid droplet canbe observed on the fourth day in 3T3-L1 after induction. It became single lipid dropletfrom the ring’s shape finally. Differentiation of 3T3-L1 was induced by Cocktail method.Intracytoplasmic lipids were quantitated by Oil Red O staining and GPDH activity wasassayed spectrophotometrically in cytosolic extracts of adipocytes by measuring theoxidation rate of NADH. The result indicated that intracytoplasmic lipids were increasedwith the differentiation time increased, and increased quickly after six days whendifferentiation medium was added. GPDH activity appeared the same regularity whichincreased quickly after four days when differentiation medium was added. The maximumvalue of GPDH activity was achieved till eight days and maintained the high level. 2. Effect of AngⅡon proliferation of 3T3-L1The proliferation of 3T3-L1 was assayed by MTT with serum and starvation method.The result indicated that AngⅡhas no significant influence on proleferation of 3T3-L1 at0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L（P＞0.05）. It suggested that AngⅡhas nosignificant influence on proliferation of 3T3-L1.3. Effect of AngⅡon differentiation of 3T3-L1 preadipocyteWe investigated changes of intracytoplasmic lipids content and GPDH activity aftertreated with AngⅡ（1μmol/L、100 nmol/L、10 nmol/L、1 nmol/L、0.1 nmol/L） accordingto its physiological concentration. AngII significantly increased lipid content at 100nmol/L, as well 10 nmol/L, and GPDH activity at 100 nmol/L over control levels by 62%,21%, 64% after 8 d. These results suggested that AngⅡpromoted the differentiation of3T3-L1 preadipocyte.4. Effect of AngⅡon the expression of transcription factors PPARγ、C/EBPα、SREBPlc mRNA during 3T3-L1 preadipocyted differentiationRT-PCR assays showed that AngⅡ（100 nmol/L） increased the expression of PPARγ、C/EBPα、SREBPlc mRNA over control levels by 48%, 50%, 43% after 6 d, consistentwith rise in lipids content and GPDH activity upon AngⅡtreatment. These findingssuggested that molecule regulation mechanisms of the positive role of AngⅡin 3T3-L1differentiation related to upregulation of expression of the PPARγ、C/EBPα、SREBPlcmRNA as adipogenic transcription factors.更多还原