【摘要】 目的:构建人Trim22的重组逆转录病毒载体,观察过表达Trim22对脂多糖(LPS)诱导的巨噬样细胞促炎细胞因子产生的影响。方法:经PCR法扩增,把Trim22克隆入逆转录病毒载体MSCV2.2 IRES-GFP(MSCV),并对重组载体进行菌落PCR、双酶切及测序鉴定。用Lipofectamine将MSCV、GAG-POL、VSV-G载体共转染至293T包装细胞。用病毒上清感染U937细胞,通过流式细胞仪分选GFP阳性细胞。用佛波酯诱导U937细胞分化为巨噬样细胞,LPS刺激后观察过表达Trim22对促炎细胞因子表达的影响。结果:经测序等鉴定,成功构建MSCV-Trim22逆转录病毒表达载体。病毒上清感染U937细胞后,经流式细胞仪分选获得稳定表达Trim22的U937细胞。LPS刺激巨噬样细胞后,Trim22过表达组TNFα和IL6的表达水平显著小于对照组(P<0.05)。结论:成功构建人Trim22的逆转录病毒表达载体,Trim22能抑制LPS诱导的巨噬样细胞TNFα和IL6的产生。更多还原

【Abstract】 Objective: To construct recombinant retrovirus vectors carrying human Trim22 gene,which could be stably expressed in U937 cell line,and to study the effects of Trim22 on pro-inflammatory cytokines production in LPS-stimulated macrophage-like cells.Methods: Trim22 gene was amplified with PCR and subcloned into retroviral vector(MSCV2.2 IRES-GFP).The recombinant vector was confirmed by restriction enzyme digestion,colony PCR and sequencing.The retroviral vector together with GAG-POL and VSV-G helper vectors were co-transfected into the packaging cells 293T by Lipofectamine.The supernatant of 293T cells was used to infect U937 cells and GFP positive U937 cells stably expressing Trim22 were sorted by flow cytometry.Eventually,PMA was used to differentiate U937 cells into macrophages and then LPS-induced pro-inflammatory cytokine levels were measured by ELISA.Results: Trim22 gene was successfully constructed into retrovirus vectors MSCV2.2 IRES-GFP,and the cloning site and reading frame were confirmed by enzyme digestion,colony PCR and sequencing.U937 cells stably expressing Trim22 and GFP were obtained by flow cytometry and were revealed by RT-PCR.Ectopic expression of Trim22 severely attenuated TNFα,IL6 production in LPS-stimulated macrophages derived from U937 cells.Conclusion: Trim22 gene retroviral vector was successfully constructed,and Trim22 could down-regulate LPS-mediated TNF-α,IL6 expression in macrophages-like cells.更多还原