Tet-off系统调控人IFNγ基因在小鼠骨髓基质细胞中的表达

Regulatory Expression of Human Interferon-gamma Gene in Murine Bone Marrow Stromal Cells by Tet-off System

背景与目的:Tet-off系统是一个高效、低毒、具有严密开关功能的基因调控系统,本研究构建质粒pTre-IFNγ,初步探讨Tet-off系统调控人干扰素γ(interferon-gamma,IFNγ)基因在小鼠骨髓基质细胞中的表达。方法:SacⅡ、XbaⅠ双酶切载体pTre及目的基因,T4连接酶连接,重组体经双酶切、测序鉴定;脂质体介导pTet-off及pTre-IFNγ共转染小鼠骨髓基质细胞;RT-PCR法检测IFNγmRNA,ELISA法检测分泌到培养液中的IFNγ蛋白,分析共转染后IFNγ的分泌规律、共转染后48h中不同浓度盐酸四环素对IFNγ蛋白分泌的影响及在共转染后第48h加入盐酸四环素在不同时间阶段对IFNγ蛋白分泌的影响。结果:双酶切及测序证实质粒pTre-IFNγ构建成功;共转染后48hRT-PCR检测到IFNγmRNA表达;ELISA法示IFNγ蛋白分泌持续6天,第3天的分泌高峰为每1×106个细胞(720.09±241.51)pg;共转染后48h中蛋白分泌量随四环素浓度增加逐渐减少,10～100ng/ml四环素存在时蛋白分泌接近零;第48h时添加盐酸四环素后8h即能明显抑制IFNγ蛋白表达,延长作用时间,抑制效应加重。结论:Tet-off系统能迅速而高效地下调人IFNγ基因在小鼠骨髓基质细胞中的表达。

BACKGROUND ＆ OBJECTIVE： The Tet-off system is a gene expression regulating system, which has high efficiency, low toxicity and strict turning-off function. This study was to investigate the regulatory expression of human interferon-gamma （IFNγ） gene in murine bone marrow stromal cells （BMSCs） by Tet-off system. METHODS： Plasmid pTre and human IFNγ gene were digested using Sac Ⅱ and Xba Ⅰ , purified, and ligated by T4 ligase. The recombinant pTre-IFNγ was identified by restriction analysis and sequencing, pTet-off and pTre-IFNγ were co-transfected into murine BMSCs. Reverse transcription-polymerase chain reaction （RT-PCR） was used to detect IFNγ mRNA 48 h after transfection. ELISA was used to detect IFNγ protein in the cell culture medium everyday to analyze the secretary mode of IFNγ. Then, Tetracycline was added to the culture medium immediately after co-transfection in gradient concentration and 48 h after cotransfection to observe its effect on IFNγ secretion. RESULTS. Restriction analysis and sequencing confirmed the orientation and sequence of the recombinant plasmid pTre-IFNγ were correct. RT-PCR detected IFNγ mRNA in BMSCs 48 h after co-transfection. ELISA showed the secretion of IFNγ lasted 6 days and a peak appeared in the third 24 h, which was （720.09± 241.51 ） pg per 1× 10^6 cells. Increasing tetracycline decreased the secretion of IFNγ in the first 48 h：10-100 ng/ml tetracycline decreased the secretion to nearly 0. When tetracycline was added 48 h after co-transfection, the secretion was obviously suppressed 8 h later, and the suppression was strengthened as time went by. CONCLUSION： The Tet-off system can efficiently and rapidly clown-regulate the expression of human IFNγ gene in murine BMSCs.