人骨形态蛋白-2可调控系统在大鼠骨髓间充质干细胞的表达

Construction of lentiviral vector containing regulatory system and bone morphogenetic protein-2 for bone marrow mesenchymal stem cells transfection

目的构建携带四环素真核诱导表达系统(Tet-on)调控的人骨形态发生蛋白2(h BMP-2)慢病毒载体转染大鼠骨髓间充质干细胞(BMSCs),为骨缺损修复提供可控的成骨活性细胞。方法设计目的基因h BMP-2引物,将扩增纯化的目的基因h BMP-2定向克隆至携带Tet-on的慢病毒GV347载体上并测序鉴定产物。h BMP-2-GV347质粒、空载的GV347质粒分别与辅助质粒共感染293T细胞以收获慢病毒浓缩液。用h BMP-2-GV347慢病毒转染大鼠BMSCs,得到h BMP-2阳性表达细胞,探索转染最佳感染复数(MOI)。用CCK8法比较BMSCs转染前后的增殖活性;强力霉素(DOX)诱导打开Tet-on,real-time PCR、Western Blot和酶联免疫吸附试验(ELISA)检测转染后各组BMSCs中BMP-2蛋白及RNA的表达情况。结果 PCR后得到目的基因h BMP-2,定向克隆至携带Tet-on系统调控的GV347质粒上,经酶切后电泳、测序结果证实成功构建携带可调控系统的h BMP-2-GV347质粒。h BMP-2-GV347质粒与空载GV347质粒分别感染293T细胞后获得h BMP-2-GV347慢病毒浓缩液。在DOX诱导下,h BMP-2-GV347慢病毒载体在293T细胞内表达BMP-2蛋白。h BMP-2-GV347慢病毒载体转染最佳MOI值为9,且转染后的BMSC细胞增殖能力较普通BMSC强,并在DOX浓度为10μg/ml诱导下稳定表达BMP-2蛋白和RNA。结论本研究成功构建携带Tet-on系统调控h BMP-2慢病毒表达载体,转染BMSCs后在DOX调控下可持续高效表达BMP-2蛋白。

Objective To construct a lentiviral vector with tetracycline regulated inducible transcriptional activation （Tet-on）system-regulated human bone morphogenetic protein-2 （hBMP-2）,and to measure the expression of hBMP-2 gene under the induction of doxycycline （ DOX） after transfection with bone marrow-derived mesenchymal stem cells （BMSCs）.Methods A lentiviral vector expressing hBMP-2 with an controllable system was constructed .The primer of target gene hBMP-2 was designed ,and the target gene hBMP-2 was directly cloned to GV347 vector with Tet-on regulatory system after PCR amplification and purification ,followed by the verification of the construction of hBMP-2-GV347 plasmid by enzyme digestion ,electrophoresis and sequencing .After transformation and amplification ,hBMP-2-GV347 plasmid and helper plasmids were co-transfected into 293T cells;after collection of lentiviral vectors ,the lentiviral vectors were transfected into 293T cells,and the intracellular expression of target gene with or without DOX was detected by Western Blot . Positive and negative BMSCs were obtained through transfection with hBMP-2-GV347 lentiviral vector and empty GV347 lentiviral vector.The best multiplicity of infection （ MOI） was tested;with or without DOX induction ,the cell proliferative activity of BMSCs before and after transfection were also examined;real-time PCR,Western Blot and enzyme-linked immunosorbent assay （ ELISA ） were applied to determine the protein and RNA expression of BMP-2 in BMSCs in BMSCs after transfection .Results The target gene hBMP-2 was obtained ,and directly cloned to linearized GV347 plasmid with Tet-on regulatory system;results of enzyme digestion ,electrophoresis and sequencing confirmed that hBMP-2-GV347 plasmid with an conrollable system was successfully constructed .Under the induction of DOX ,hBMP-2-GV347 lentiviral vector expressed BMP-2 protein in 293T cells.As for the transfection with hBMP-2-GV347 lentiviral vector ,the optimum MOI value was 9;after transfection ,the cell proliferation ability of transfected BMSCs was stronger than that of common BMSCs,and transfected BMSCs continuously and stably expressed BMP-2 protein with high mRNA level under the induction of DOX （10 μg /ml）.Conclusion A lentiviral vector with Tet-on system-regulated hBMP-2 has been succefully constructed and the transefected BMSCs can provide controllable BMP -2 secretion for bone defect repair .