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目的观察NYGGF4基因过表达对3T3-L1脂肪前体细胞增殖的影响,探讨该基因在肥胖发生发展中的作用。方法采用反转录聚合酶链反应(RT-PCR)法扩增NYGGF4开放阅读框(ORF)的全长,通过双酶切连接法构建NYGGF4-pcDNA3.1真核表达载体。采用脂体法转染3T3-L1前体脂肪细胞,以pcDNA3.1空载和未转染细胞为对照。遗传霉素(G418)压力筛选稳定转染细胞株,PCR鉴定转染细胞的NYGGF4的mRNA水平。采用四甲基偶氮唑蓝(MTT)检测稳定转染细胞株连续7 d的生长状态,以转染空载和未转染正常3T3-L1为对照,绘制生长曲线。采用SPSS 10.0软件进行统计学分析。结果1.稳定转染细胞株有NYGGF4基因的表达,而pcDNA3.1空载未见人源性NYGGF4的表达;2.转染NYGGF4基因的3T3-L1细胞增殖速度明显较未转染和转染空载的3T3-L1细胞加快。结论新基因NYGGF4可明显促进3T3-L1脂肪前体细胞的增殖,可能影响肥胖的发生发展。
Objective To observe the effect of NYGGF4 gene overexpression on the proliferation of 3T3-L1 adipose precursor cells and to explore its role in the development of obesity. Methods The full length of NYGGF4 open reading frame (ORF) was amplified by reverse transcription polymerase chain reaction (RT - PCR). The eukaryotic expression vector NYGGF4 - pcDNA3.1 was constructed by double enzyme digestion. Liposome-transfected 3T3-L1 preadipocytes were transfected with pcDNA3.1 vector and untransfected cells as control. Genomicin (G418) was used to screen stable transfected cell lines by pressure screening. The mRNA level of NYGGF4 in transfected cells was identified by PCR. The growth of stably transfected cells was detected by MTT assay. The growth curve was drawn by transfecting the normal and untransfected 3T3-L1 cells. Using SPSS 10.0 software for statistical analysis. Results 1. Stably transfected cell lines had NYGGF4 gene expression, but pcDNA3.1 no human expression of NYGGF4 empty; 2. Transfection of NYGGF4 gene 3T3-L1 cells were significantly faster than untransfected and transfected The empty 3T3-L1 cells are accelerated. Conclusion The new gene NYGGF4 can significantly promote the proliferation of 3T3-L1 adipose precursor cells, which may affect the occurrence and development of obesity.