摘要
目的观察甘油醛-3-磷酸脱氢酶（GAPDH）在3T3-L1脂肪细胞分化过程中表达水平是否存在变化，并与其他常用的内参基因相比较。方法以实时定量PCR检测3T3-L1脂肪细胞分化0、1、3、5、7d几种不同常见内参基因的表达是否存在变化，并以Western印迹方法进行证实。结果（1）内参基因GAPDH和转铁蛋白受体（TFRC）在脂肪细胞分化过程中基因表达水平逐渐明显升高，其中GAPDHmRNA在脂肪细胞分化1、3、5、7d分别增加5．7、7．6、22．0和24．5倍（均P〈0．01），β-actin、α-微管蛋白（d—tubuhn）、肽酰脯氨酰异构酶（PIPA）和18SmRNA表达水平未见明显改变；采用实时定量PCR检测脂肪细胞分化的关键转录因子PPARγ2、CCAAT／增强结合蛋白（C／EBP）d和C／EBPβ的表达时，以GAPDH作内参明显低估他们的表达变化；GAPDH蛋白表达也随着脂肪细胞分化逐渐增加，β—actin、α-tubulin蛋白表达未见明显变化：（2）小檗碱明显抑制脂肪细胞分化过程中GAPDH mRNA和蛋白的表达，在脂肪细胞分化5、7d时GAPDH mRNA表达水平分别降低68．1％和66．3％（P〈0．05或P〈0．01），但小檗碱对其他内参基因的表达无明显改变。结论GAPDH在3T3-L1脂肪细胞分化过程中表达增加，不适合作为内参。
Objective To observe the change of glyceraldehyde-3-phosphate dehydrogenase （GAPDH） gene expression during 3T3-L1 adipocyte differentiation as well as other reference gene expressions. Methods The nlRNA expressions of several common reference genes were detected by real time-PCR on day 0, 1, 3, 5, and 7 of 3T3-L1 adipocyte differentiation. Western blot was used to confirm the protein expressions of three common reference genes. Results （ 1 ） GAPDH and transferrin receptor （TFRC） mRNA expressions were significantly increased during adipocyte differentiation. GAPDH mRNA level was increased by 5.7,7.6,22.0, and 24.5 folds on day 1,3,5, and 7 after induction of adipoeyte differentiation, but no apparent changes of β-actin, α-tubulin, peptidylprolyl isomerase A （PIPA） , and 18S mRNA expressions were detected. The expression changes of key transcript factors for adipocyte differentiation such as PPAR＇y2, C/EBPc~, and C/EBP[3 were under-estimated by real time-PCR if GAPDH was chosen as the reference gene. Western blotting results showed that the GAPDH protein level increased gradually during adipocyte differentiation, especially on day 5 and 7 after adipocyte differentiation. There were no obvious changes of β- aetin and α-tubulin protein expressions. （2） Berberine significantly inhibited mRNA and protein expressions of GAPDH in the process of adipocyte differentiation. GAPDH mRNA levels were reduced by 68.1% and 66.3% on day 5 and 7 after induction of adipocyte differentiation, but with no significant change in other reference genes. Conclusion It is not suitable for GAPDH to be used as an endogenous reference gene during 3T3-L1 adipocyte differentiation.
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2012年第10期849-854,共6页
Chinese Journal of Endocrinology and Metabolism
基金
国家自然科学基金项目（81070652、81070617、81170720）
关键词
甘油醛-3-磷酸脱氢酶
内参基因
脂肪细胞分化
小檗碱
Glyeeraldehyde-3-phosphate dehydrogenase
Reference gene
Adipocyte differentiation
Berberine
