代综方对3T3-L1脂肪细胞分化及相关基因表达的影响

Effects of Dai Zong Fang on 3T3-L1 adipocyte differentiation and related gene expression

摘要:

目的：研究代综方（Dai Zong Fang，DZF）对3T3-L1脂肪细胞分化及分化过程中相关基因表达的影响。方法：体外培养3T3-L1前脂肪细胞，鸡尾酒式诱导脂肪细胞分化，给予不同浓度DZF干预6 d。BODIPY493/503与DAPI双荧光染色观察脂肪细胞分化情况；甘油磷酸氧化酶-过氧化物酶法测定细胞内甘油三酯（triglyceride，TG）含量；RT-PCR检测CCAAT增强子结合蛋白-α（CCAAT enhancer binding protein-α，C/EBP-α）、过氧化物酶体增殖物激活受体-γ（peroxisome proliferator activated re－ceptor-γ，PPAR-γ）、乙酰辅酶A 羧化酶（acetyl-CoA carboxylase，ACC）、脂肪酸合成酶（fatty acid synthetase，FAS）的mRNA表达；Western blot法检测PPAR-γ、C/EBP-α的蛋白表达。结果：DZF抑制脂肪细胞分化，降低细胞内TG含量，并呈剂量依赖效应；与对照组比较，DZF大剂量组明显下调ACC的mRNA表达（P<0.05），DZF小中大剂量均明显下调FAS的mRNA表达（P<0.01）；与对照组比较，DZF中、大剂量组明显下调C/EBPα、PPAR-γ的mRNA表达（P<0.01），Western蛋白表达结果与mRNA结果一致。结论：DZF能够明显抑制3T3-L1脂肪细胞的分化程度，其机制可能与下调C/EBPα、PPAR-γ、ACC、FAS基因的表达有关。

Abstract:

Objectives：To study the effect of Dai Zong Fang（DZF） on 3T3-L1 preadipocytes differentiation and changes in expression of differentiation associated genes. Methods：3T3-L1 preadipocytes cells were induced into mature adipocytes by Cocktail（1-methyl-3-isobutylxanthine，dexamethasone and insulin），treated by different concentrations of DZF from 2 d to 8 d of differentiation. The lipid accumulation of adipocyte was observed by BODIPY493/503 and DAPI double fluorescent staining. The intracellular triglyceride（TG） was detected by glycerol phosphate oxidase and peroxidase. RT-PCR was used to detect mRNA expression of CCAAT enhancer bind－ing protein-α（C/EBP-α），peroxisome proliferators-activated receptors-γ（PPAR-γ），acetyl-CoA carboxylase（ACC），fatty acid syn－thetase（FAS）. Western blot was used to detect protein expression of PPAR-γ and C/EBP-α. Results：DZF could inhibit adipocyte differentiation and reduce intracellular TG content in a dose-dependent manner. ACC mRNA expression was significantly down-regulated in large-dose DZF group than in control group（P<0.05）. FAS mRNA expression was significantly down-regulated in small，medium，large-dose DZF groups than in control group（P<0.01）. C/EBPα and PPAR-γ mRNA expression was significantly down-regulated in medium，large-dose DZF groups than in control group（P<0.01）. The result of Western blot was consistent with that of mRNA results. Conclusion：DZF could significantly inhibit the differentiation of adipocyte，the mechanisms might be related to the down-regulation of C/EBPα，PPAR-γ，ACC，FAS gene expression.