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目的构建高表达磷脂转运蛋白的细胞模型,探讨半枝莲总黄酮对磷脂转运蛋白(PLTP)表达的影响。方法利用RT-PCR方法扩增磷脂转运蛋白(PLTP)cDNA片段,将其克隆到p3XFLAG-CMV-14载体,得到重组载体PLTP/p3XFLAG-CMV-14,转染于人肝癌细胞系Bel-7402。以不同浓度的半枝莲总黄酮作用于重组细胞,用Western blotting和免疫细胞化学方法检测细胞中PLTP的表达变化。结果转染的人肝癌细胞系Bel-7402细胞模型中PLTP的表达显著增强;与模型(转染于人肝癌细胞系Bel-7402)组比较,终浓度为160 mg/L的半枝莲总黄酮干预Bel-7402细胞24h后,差异有显著性(P<0.05);48h时差异非常显著(P<0.01);72h时差异极显著(P<0.001)。与阳性对照组比较,半枝莲总黄酮高、中、低3个剂量下,作用24、48、72h均无显著性差异(P>0.05)。结论应用RT-PCR、基因重组技术构建了基因重组细胞模型Bel-7402;半枝莲总黄酮可显著抑制重组Bel-7402细胞PLTP的表达。
Objective To construct a cell model with high expression of phospholipid transporter and explore the effect of total flavonoids of Scutellaria barbata on the expression of phospholipid transporter (PLTP). Methods The fragment of phospholipid transfer protein (PLTP) was amplified by RT-PCR and cloned into p3XFLAG-CMV-14 vector. The recombinant plasmid PLTP / p3XFLAG-CMV-14 was obtained and transfected into human hepatocellular carcinoma cell line Bel-7402. The effects of different concentrations of Scutellaria barbata total flavone on the expression of PLTP in cells were detected by Western blotting and immunocytochemistry. Results Compared with the model group (Bel-7402 cells transfected with human hepatocellular carcinoma cell line), the expression of PLTP in the transfected human hepatocellular carcinoma cell line Bel-7402 was significantly enhanced. Compared with the control group, the total flavonoids of Scutellaria barbata The difference was significant at 24 h (P <0.05). The difference was significant at 48 h (P <0.01). The difference was significant at 72 h (P <0.001). Compared with the positive control group, Scutellaria barbata total flavonoids at high, medium and low doses of 3, the role of 24,48,72 h had no significant difference (P> 0.05). Conclusion The recombinant plasmid Bel-7402 was constructed by RT-PCR and gene recombination technique. Total flavonoids of Scutellaria barbata D. Don could significantly inhibit the expression of PLTP in recombinant Bel-7402 cells.