摘 要

肥胖是由于脂肪细胞数目增多及体积增大而导致过多的白色脂肪组织(WAT)在体内积累。脂肪形成过程实际上是脂肪组织中的细胞经过增殖、分化形成可以累积脂质细胞的过程。有多种转录因子调控这一过程。3T3-L1 前脂肪细胞系是建立成熟脂肪细胞模型最为常用的细胞系之一。前期实验表明银杏叶提取物（EGB）活性成分银杏内酯处理对小鼠脂肪细胞（3T3-L1）中一些脂肪代谢的调控因子的mRNA表达有显著影响及其可能的作用机理。油红O染色法检测表明银杏内酯对前脂肪细胞分化和脂质积聚有一定的抑制效果。使用诱导剂对 3T3-L1 前脂肪细胞进行诱导分化。本实验拟在分化过程中用银杏内酯进行给药处理， 收集分化到第八天的3T3-L1细胞，提取RNA并逆转录为cDNA。通过实时荧光定量PCR的方法检测两种银杏内酯单体处理过的3T3-L1细胞中调控脂肪代谢的细胞因子，酶或标记蛋白的mRNA表达水平，结果表明一定浓度的银杏内酯单体处理能够降低与脂肪合成相关的基因mRNA表达水平，并在一定程度上提升与脂肪降解相关的基因mRNA表达水平，并改变与脂肪代谢相关的转录因子的mRNA表达水平，这在分子水平上为实验猜想提供了依据。从中选出有显著变化的，为今后在蛋白质水平上进一步验证，推测并证实其信号传导途径提供依据，以期为肥胖等代谢性疾病的治疗提供一定的理论指导和新的治疗思路。

关键词：3T3-L1；前脂肪细胞分化；脂肪代谢；转录因子；脂肪代谢酶

Effect of Ginkgolides on mRNA Expression of Several Regulatory Genes Affecting Fat Metabolism in Mouse Adipocytes

ABSTRACT

Obesity is caused by excessive accumulation of white adipose tissue (WAT) in the body. The process of fat formation is actually a process of proliferation and differentiation from preadipocyte to mature adipocyte, resulting in the increased number and volume of adipocytes,. The 3T3-L1 preadipocyte cell line is one of the most commonly used cell lines for establishing a mature adipocyte model. Previous experiments showed that the treatment of Ginkgo biloba extract (EGB) had a significant effect on mRNA expression of some lipid metabolism regulators in 3T3-L1 cells. Also Oil red O staining result showed ginkgolide A and B had certain inhibition effect on differentiation and lipid accumulation in differentiating 3T3-L1. This study was to evaluate several genes that regulate lipid metabolism by detecting the mRNA expression in 3T3-L1 cells treated with various concentration of ginkgolide A and B via real-time quantitative PCR. The results showed that a certain concentration of ginkgo lactone monomer treatment can reduce the expression level of mRNA related to fat synthesis, and increase the expression level of mRNA related to fat degradation to a certain extent, and change the mRNA of transcription factor related to fat metabolism. The level of expressionprovides a basis for experimental conjecture at the molecular level.The protein expression will be further verified by western blotting for those with significant changes in mRNA expression level. The signal transduction pathways will be confirmed, to provide basis for the mechnism study and new therapeutic ideas for the treatment of obesity.

Key words：3T3-L1; Preadipocyte Differentiation; Lipid Metabolism; Transcription Factor; Lipid Metabolizing Enzyme

目录

1文献综述………………………………………………………………………………………5

1.1简介…………………………………………………………………………………………5

1.2脂肪代谢……………………………………………………………………………………5

1.2.1脂肪细胞分化…………………………………………………………………………5

1.2.2代谢过程………………………………………………………………………………6

1.2.3调控基因对脂肪代谢的影响…………………………………………………………6

2材料与方法………………………………………………………………………………………7

2.1材料………………………………………………………………………………………7

2.1.1主要试剂………………………………………………………………………………7

2.1.2仪器设备……………………………………………………………………………7

2.2方法……………………………………………………………………………………………7

2.2.13T3-L1 前脂肪细胞的培养……………………………………………………………7

2.2.2MTT法检测3T3-L1 前脂肪细胞增殖…………………………………………………8

2.2.3试剂盒法提取RNA………………………………………………………………………8

2.2.4测RNA浓度……………………………………………………………………………9

2.2.5逆转录成cDNA…………………………………………………………………………9

2.2.6qPCR法用不同引物扩增片段…………………………………………………………10

1. 结果与分析……………………………………………………………………………………12
2. 结论……………………………………………………………………………………………15
3. 致谢……………………………………………………………………………………………16

参考文献……………………………………………………………………………………………17

1文献综述