Tet-off系统对小鼠骨髓基质细胞中人IFN-γ基因表达的调控作用

Regulating Human Interferon-gamma Gene Expression in Marrow Stromal Cells in Mice by Tet-off System

背景与目的:本实验室已成功构建了质粒pTre-IFN-γ,并证实四环素基因调控系统(Tet-off system)能在体外调控人γ-干扰素(interferon-gamma,IFN-γ)基因在小鼠骨髓基质细胞(marrow stromal cells,MSCs)中的表达。本实验进一步研究Tet-off系统体外调控的可逆性,以及其对小鼠MSCs中人IFN-γ基因表达的调控作用。方法:脂质体介导pTet-off和pTre-IFN-γ质粒共转染小鼠MSCs。用ELISA法检测MSCs培养液中人IFN-γ蛋白的分泌量。把共转染后的MSCs回输给BALB/c裸小鼠,用实时荧光定量RT-PCR方法检测各组BALB/c裸小鼠脾组织中的IFN-γmRNA含量。结果:用ELISA法可检测到共转染后的MSCs培养液中有IFN-γ蛋白分泌,分泌高峰在前72h内;共转染后84h加入含300ng/mL盐酸四环素的培养液培养12h,每1×107个细胞的分泌量为(67.11±22.14)pg,无四环素处理组为(319.96±29.04)pg,两组相比差异有统计学意义(P<0.001);去除四环素后,IFN-γ蛋白分泌显著增加(P=0.032)。用实时荧光定量RT-PCR可检测到回输共转染MSCs的各组BALB/c裸小鼠脾组织中均有IFN-γmRNA转录,不用四环素处理组最高,达(1.5±0.7)×105copy·(100mg)-1,定期四环素处理组为(6.9±5.3)×102copy·(100mg)-1(P<0.001),接受1次四环素处理组表达量介于前两者之间,差异均有统计学意义(P<0.01)。结论:Tet-off系统在体外和体内都可以迅速、有效地调控人IFN-γ基因在小鼠MSCs中的表达,而且调控是可逆的。

BACKGROUND ＆ OBJECTIVE. We have constructed plasmid “pTre-IFN-γ” and proved that the Tet-off system could regulate the expression of human interferon-gamma （IFN-γ） gene in murine marrow stromal cells in vitro. This study was to investigate the regulatory reversibility of Tet-off system and its effect on the expression of human IFN-γ gene in murine marrow stromal cells in mice. METHODS： Plasmids pTet-off and pTre-IFN-γ were cotransfected into mudne marrow stromal cells. The expression of IFN-γ in marrow stromal cells was detected with ELISA. The marrow stromal cells were transfused into BABL/c naked mice after co-transfection. The expression of IFN-γ mRNA in the spleen was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： IFN-γ protein was detected in the culture solution of marrow stromal cells after cotransfection. The secretion peak appeared within the first 72 h. The protein level of IFN-γ was significantly lower in 300 ng/ml tetracycline hydrochloridetreated marrow stroma cells than in untreated cells [（67.11±22.14） pg/1×10^7 cells vs. （319.96±29.04） pg/1×10^7 cells, P〈0.001]; its expression was increased when removed tetracycline hydrochloride （P=0.032）. The expression of human IFN-γ mRNA was detected in the spleen. The mRNA level of IFN-γ was significantly higher in untreated group than in continuous tetracycline hvd rochloride-treated group L- （ 1.5±0.7 ） ×10^-1 copies. （ 100 mg ）^-1 vs. （6 9±5.3） ×10^2 copies·（100 mg）^-1, P〈0.001]; its expression in the mice received tetracycline hydrochloride for one single time lay between the above two groups with significant difference. CONCLUSION： In mice, Tet-off system could rapidly, efficiently and reversibly regulate the expression of human IFN-γ gene in marrow stromal cells in vitro and in vivo.