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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 the important cytokines that mediate the maturation of dendritic cells. Soluble tumor necrosis factor receptor-1 binds to it and blocks the effect of tumor necrosis factor-α. It maintains dendritic cells in an immature state, Induction of immune tolerance. Objective: To construct a lentiviral vector containing human sTNFR1 and observe its expression in immature dendritic cells. Methods: The total RNA of human peripheral blood mononuclear cells was used as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned into lentiviral plasmid pXZ208, and eGFP reporter gene was linked by IRES to construct bicistronic lentiviral vector. Named pXZ9-sTNFR1, DNA sequencing identification. 293FT cells were transfected with liposomes and the virus titer was determined by eGFP. Bone marrow-derived dendritic cells from C57BL / 6 mice were cultured in vitro with low-dose granulocyte-macrophage colony-stimulating factor + interleukin-4. On day 5 of culture, immature dendritic cells were infected with pXZ9-sTNFR1 recombinant lentivirus supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression was detected by Western blot, and sTNFR1 gene modification and dendritic stimulated Phenotypic characteristics of somatic cells. RESULTS AND CONCLUSION: The recombinant plasmid pXZ9-sTNFR1 was successfully constructed and eGFP expression was observed at 24h after transfection in 293FT cells. The virus titer was above 106U / L. RT-PCR showed that sTNFR1 of immature dendritic cells infected by pXZ9-sTNFR1 was positive, and sTNFR1 protein was detected by Western blot in immature dendritic cells and culture supernatant after infection. Dendritic cells cultured on day 5 showed low expression of CD40, CD86, CD80 and MHC class II molecules. After stimulation with lipopolysaccharide, MHC class II molecules and CD40, CD80 and CD86 molecules were highly expressed, showing the phenotypic characteristics of mature dendritic cells The expression of MHC class II molecules and CD40, CD80 and CD86 molecules of dendritic cells modified by sTNFR did not change. Tip: ① successfully constructed sTNFR1 gene fragment and lentiviral vector containing eGFP reporter gene, obtained a high titer of recombinant lentiviral particles. (2) Stable expression of sTNFR1 mRNA and protein in immature dendritic cells highly transduced by lentivirus can protect immature dendritic cells from stimulation by exogenous lipopolysaccharide and maintain dendritic cells in a non-mature state.