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目的探讨利用Tet-on基因表达系统调控目的基因在骨髓基质干细胞中表达的可能性和量效关系,为利用该系统调控BMP基因在骨髓基质干细胞中的表达奠定基础。方法以荧光素酶(luciferase)基因为目的基因,应用Lipofectamine介导的瞬间转染方法,将pTet-on调节质粒和pTRE-luciferase反应质粒共转染骨髓基质干细胞,加入系列浓度的强力霉素(doxycyline)后,观察doxy-cyline的诱导效应。结果(1)pTet-on表达系统质粒DNA经HindⅢ单酶切、琼脂糖凝胶电泳后,可见6.02kb和1.38kb两个片段;pTRE-luciferase质粒经HindⅢ单酶切线性化,可见5.2kb的片段,均符合质粒图谱。(2)随着doxycyline浓度的增加,荧光素酶的活性也逐渐增加。未转染的骨髓基质干细胞虽经doxycyline诱导,但其RLU值与空白对照(蒸馏水)非常接近,说明未转染的骨髓基质干细胞不受doxy-cyline诱导。而转染的骨髓基质干细胞在未加入诱导剂时,其RLU(relative-luciferase-unit)值也接近空白对照值。结论转染Tet-on基因表达控制系统的骨髓基质干细胞对doxycyline诱导具有较好的反应性,目的基因荧光素酶基因的表达效力对doxycyline具有严格的计量依赖关系。因此,利用Tet-on基因调控系统对目标基因在骨髓基质干细胞中的表达进行调控是可行的。
OBJECTIVE: To investigate the possibility and dose-effect relationship of using Tet-on gene expression system to regulate the expression of target gene in bone marrow stromal stem cells, and to lay a foundation for the regulation of BMP gene expression in bone marrow stromal cells by this system. Methods The luciferase gene was transfected into bone marrow stromal cells using pTet-on regulatory plasmid and pTRE-luciferase reaction plasmids by Lipofectamine-mediated transient transfection. A series of concentrations of doxycycline doxycyline) to observe the induction effect of doxy-cyline. Results (1) The pTet-on expression system plasmid DNA by Hind Ⅲ single digestion, agarose gel electrophoresis, visible 6.02kb and 1.38kb two fragments; pTRE-luciferase plasmid by HindIII single digestion linearization, we can see 5.2kb Fragments, all in line with the plasmid map. (2) With the increase of doxycyline concentration, luciferase activity also increased gradually. The untransfected bone marrow stromal cells were induced by doxycyline, but their RLU values were very close to that of blank control (distilled water), indicating that untransfected bone marrow stromal cells were not induced by doxy-cyline. The transfection of bone marrow stromal cells RLU (relative-luciferase-unit) RLU value is also close to the blank control value. CONCLUSION: Bone marrow stromal stem cells transfected with Tet-on gene expression control system have good reactivity to doxycyline induction. The expression of target gene luciferase gene has a strict dose-dependent relationship with doxycyline. Therefore, it is feasible to use Tet-on gene regulation system to regulate the expression of target gene in bone marrow stromal stem cells.