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目的:为探讨对氧磷酯酶1(PON1)在机体代谢性疾病中的作用及机制,本实验构建含人源性PON1基因的慢病毒表达载体,研究其表达分泌PON1的能力及对小鼠巨噬细胞的影响。方法:根据人肝cDNA文库的PON1序列设计特定引物,定点诱变PCR获得两端为PmeⅠ内切酶位点的目的基因,经酶切连接到pWPI-GFP载体上,筛选及DNA测序鉴定。磷酸钙法瞬时转染293T细胞,荧光显微镜观察GFP表达反映转染效率,Western Blotting检测细胞培养基中PON1分泌表达量,采用对氧磷为底物检测PON1活性。含PON1的培养基与小鼠腹腔巨噬细胞共孵育,ELISA法检测氧化条件下细胞分泌肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)的水平。结果:构建的pWPI-PON1慢病毒表达载体序列正确,高效转染293T细胞(转染效率可达到80%-90%)可有效分泌表达PON1至培养基,显著提高培养基PON1活性,与对照组相比,培养基中PON1可明显抑制巨噬细胞炎症因子TNF-α、IL-6的分泌(P<0.01)。结论:pWPI-PON1慢病毒载体高效表达分泌的外源PON1能有效降低巨噬细胞的炎症反应。
Objective: To investigate the role and mechanism of paraoxonase 1 (PON1) in metabolic diseases of the body, we constructed a lentiviral expression vector containing human PON1 gene and studied its ability to express PON1 and its effect on mice Effects of macrophages. Methods: According to the PON1 sequence of human liver cDNA library, specific primers were designed. The target gene with Pme Ⅰ endonuclease at both ends was obtained by site-directed mutagenesis. The recombinant plasmid was ligated into pWPI-GFP vector and identified by DNA sequencing. The transient transfection of 293T cells was performed by calcium phosphate method. The expression of GFP was observed by fluorescence microscopy, and the expression of PON1 was detected by Western Blotting. The activity of PON1 was detected by using paraoxon as substrate. PON1-containing medium was co-incubated with mouse peritoneal macrophages, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secreted by cells under oxidative stress were detected by ELISA. Results: The constructed pWPI-PON1 lentiviral vector was successfully transfected into 293T cells (80% -90% transfection efficiency), which can effectively secrete PON1 into the medium and significantly increase the activity of PON1 in the medium. Compared with the control group PON1 in culture medium significantly inhibited the secretion of inflammatory cytokines TNF-α and IL-6 (P <0.01). Conclusion: The pWPI-PON1 lentiviral vector efficiently expresses secreted exogenous PON1 can effectively reduce macrophage inflammatory response.