中文摘要：

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。

英文摘要：

Objective：To explore the effect and mechanism of paraoxonase 1（PON1）in the metabolic diseases,human PON1 gene were constructed into the pWPI-GFP lentivirals vector,the expression ability after transfected into eukaryotic cells and the role of enzyme on mice macrophages were studied.Methods：PON1gene was amplified from human hepatocytes cDNA library by PCR,conjugated into PWPI-GFP vector using the restriction enzyme PmeⅠ and checked by DNA sequencing.The expression level of PON1 was detected by Western blotting after vectors were transiently transfected into 293 Tcells using the calcium phosphate method,and PON1 activity in medium was determined with paraoxon as substrate.Then mouse peritoneal macrophages were incubated for 24 hwith medium containing oxidative LDL with or without PON1,tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）were determined by ELISA.Results：Recombinant pWPI-PON1 lentivirus vector were successfully constructed and verified by DNA sequencing.PON1 was efficiency expressed in 293Tcells（the transfection efficiency was up to 80%-90%）,and secreted into the medium with significantly higher activity.Compared with control group,PON1-containing medium could attenuate the release of inflammatory cytokine TNF-αand IL-6in macrophages（P〈0.01）.Conclusion：PON1can be overexpressed and secreted using lentivial vectors,and reduce macrophages inflammatory responses.