慢病毒介导可溶性肿瘤坏死因子受体1在小鼠骨髓未成熟树突状细胞中的表达(英文)

Lentivirus-mediated soluble tumor necrosis factor receptor 1 expression in mouse bone marrow-derived immature dendritic cells

背景:肿瘤坏死因子α是介导树突状细胞成熟的重要细胞因子之一,可溶性肿瘤坏死因子受体1与其结合可阻断肿瘤坏死因子α的作用,维持树突状细胞于不成熟状态,诱导免疫耐受。目的:构建含有人sTNFR1的慢病毒表达载体,观察其在未成熟树突状细胞中的表达。方法:以人外周血单个核细胞总RNA为模板,RT-PCR扩增出sTNFR1基因片段,亚克隆至慢病毒转移质粒pXZ208,通过IRES连接eGFP报告基因,建立双顺反子慢病毒转移质粒,命名为pXZ9-sTNFR1,DNA测序鉴定。采用脂质体转染293FT细胞,根据报告基因eGFP测定病毒滴度。采用小剂量粒-巨噬细胞集落刺激因子+白细胞介素4体外培养扩增C57BL/6小鼠骨髓来源树突状细胞。培养第5天,以pXZ9-sTNFR1重组慢病毒上清感染未成熟树突状细胞,RT-PCR检测感染后sTNFR1转录,Western blot法检测sTNFR1蛋白表达,观察sTNFR1基因修饰及脂多糖刺激后树突状细胞的表型特征。结果与结论:成功构建重组质粒pXZ9-sTNFR1,转染293FT细胞24h后观察到eGFP表达,病毒滴度在106U/L以上。RT-PCR显示pXZ9-sTNFR1感染的未成熟树突状细胞sTNFR1呈阳性表达,Western blot检测到sTNFR1蛋白存在于感染后未成熟树突状细胞和培养上清中。培养第5天的树突状细胞低表达CD40、CD86、CD80和MHCⅡ类分子,脂多糖刺激后,高表达MHCⅡ类分子和CD40、CD80、CD86分子,显示出成熟型树突状细胞表型特征,sTNFR修饰的树突状细胞MHCⅡ类分子和CD40、CD80、CD86分子表达水平无变化。提示:①成功构建了负载sTNFR1基因片段及含eGFP报告基因的慢病毒载体,获得了高滴度的重组慢病毒颗粒。②经慢病毒高效转导的未成熟树突状细胞sTNFR1 mRNA及蛋白稳定地表达,可以保护未成熟树突状细胞不被外源性脂多糖刺激活化,维持树突状细胞于非成熟状态。

BACKGROUND：Tumor necrosis factor-α（TNF-α） is one of important cytokines to promote the maturation of dendritic cells.Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1（sTNFR1） may arrest dendritic cells in an immature state and induce stable,long-term tolerance.OBJECTIVE：To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells.METHODS：Total RNA of human peripheral blood mononuclear cells was taken as a template.The sTNFR1 gene fragment was amplified by RT-PCR,subcloned to the lentiviral vectors pXZ208,and ligated to the enhanced green fluorescent protein（eGFP） reporter gene to establish lentiviral vector,called pXZ9-sTNFR1.DNA sequencing was performed for lentiviral vector identification.Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1.Viral titer was determined by eGFP expression.C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4.On day 5 of culture,immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant.sTNFR1 transcription was detected by RT-PCR,sTNFR1 protein expression by Western blot analysis.Following sTNFR1 gene modification and lipopolysaccharide stimulation,the phenotype characteristics of dendritic cells were observed.RESULTS AND CONCLUSION：Recombinant plasmid pXZ9-sTNFR1 was successfully constructed.Twenty-four hours after 293 FT cell transfection,eGFP expression was observed and viral titer was over 106 U/L.RT-PCR demonstrated that pXZ9-sTNFR1-transfected immature dendritic cells showed sTNFR1 positive expression.Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection.On day 5 of culture,dendritic cells expressed low level of class Ⅱ major histocompatibility complex（MHC Ⅱ）,as well as CD40,CD86,CD80,molecules.However,following lipopolysaccharide stimulation,dendritic cells expressed high level of MHC Ⅱ,as well as CD40,CD80,and CD86,molecules,exhibiting the phenotype characteristics of mature dendritic cells.But after sTNFR modification,the expression level of MHC Ⅱ,as well as CD40,CD80,and CD86,molecules was not altered obviously.Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed,and recombinant lentiviral plasmids with high titer were acquired.Following high efficacy of lentiviral gene transfection,immature dendritic cells stably express sTNFR1 mRNA and protein,which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.