BMP-2基因重组慢病毒载体转染猪BMSCs成骨分化研究

OSTEODIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AFTER TRANSFECTED BY LENTIVIRAL VECTOR MEDIATED BONE MORPHOGENETIC PROTEIN 2

目的构建BMP-2基因重组慢病毒载体,并检测其转染猪BMSCs后BMP-2表达活性及诱导BMSCs成骨分化情况,为进一步构建组织工程骨软骨奠定基础。方法取2只2月龄巴马香猪(体重约15 kg)髂骨骨髓,采用密度梯度离心法分离培养BMSCs,并传代。利用基因重组技术构建BMP-2基因重组慢病毒载体,并以感染复数(multiplicity of infection,MOI)10、25、50、100、200转染第2代BMSCs,通过荧光显微镜及倒置相差显微镜观察确定最佳MOI值。以最佳MOI值感染BMSCs作为实验组,空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫组织化学染色、Western blot检测BMP-2 mRNA及蛋白在BMSCs中的表达,并应用ALP染色、ALP活性检测及茜素红钙结节染色检测其成骨分化情况。结果经RT-PCR基因测序鉴定BMP-2基因重组慢病毒载体构建成功,转染BMSCs后荧光显微镜下可见绿色荧光表达,且MOI值为100时为最佳表达。RT-PCR提示BMP-2基因成功转染至BMSCs中;免疫组织化学染色及Western blot检测示转染后BMSCs并可持续、稳定、高效表达BMP-2蛋白;培养后2周BMSCs可见ALP表达及钙结节形成。结论成功构建BMP-2基因重组慢病毒载体并转染猪BMSCs,BMP-2基因转染入BMSCs后能持续、稳定、高效表达BMP-2基因和蛋白,并成功诱导BMSCs向成骨细胞分化。

Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 （BMP-2） gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells （BMSCs） osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines （weighing, 15 kg）, and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection （MOI） of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group （BMP-2 vector group）; BMSCs transfected by empty vector （empty vector group）, and non-transfected BMSCs （non-transfection group） were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase （ALP） staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentivirol vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP-2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.