奶牛ADRP基因超表达细胞株的建立及对细胞脂滴的影响

Establishment of bovine ADRP overexpression cell line and its effect on lipid droplet

通过PCR方法扩增奶牛脂肪分化相关蛋白(adipose differentiation-related protein,ADRP)基因的编码区,构建Lenti-ADRP慢病毒重组载体,包装成病毒后感染奶牛乳腺上皮细胞,通过嘌呤霉素筛选获得超表达ADRP细胞株,采用Real-time PCR和Western blot分析细胞株中ADRP基因和蛋白的表达情况;采用尼罗红对脂滴染色,分析ADRP超表达对细胞脂滴的影响。结果显示,克隆的ADRP基因CDS区序列大约1 353 bp,成功构建重组的ADRP慢病毒载体,用5 mg/L嘌呤霉素筛选得到ADRP超表达乳腺上皮细胞株;与对照组细胞相比,超表达细胞株的ADRP基因mRNA水平增加632倍(P<0.001),蛋白表达也有所增加;检测脂滴的荧光值发现,ADRP超表达显著增加了细胞脂滴含量(P<0.01)。结果表明,超表达ADRP可促进乳腺上皮细胞的脂滴合成。

Coding region of bovine ADRP(adipose differentiation-related protein) gene was amplified by PCR,and Lenti-ADRP lentivirus recombinant vector was constructed.The virus was packaged into recombinant vector and infected dairy mammary epithelial cells.ADRP overexpression cell lines were selected by applying puromycin in culture medium.Expression of ADRP gene and protein of cell lines was detected by real-time PCR and Western blot.Effect of ADRP overexpression on cell lipid droplets was analyzed by Nile red staining.The results showed that the CDS sequence of the cloned ADRP gene was about 1 353 bp.The lentiviral vector recombinant with ADRP was successfully constructed.ADRP cell line was selecteded by 5 mg/L purinomycin.Compared with control group,the expression level of ADRP gene mRNA was 632 times higher(P<0.001) and the protein expression was also increased in overexpression ADRP cell line.Fluorescence value of lipid droplets showed that ADRP overexpression significantly increased the content of lipid droplets(P<0.01).In this study,overexpression of ADRP promoted lipid droplet synthesis in mammary epithelial cells.