Xu Shenggui, Lin Jianhua, Liu Weinan, Wu Chaoyang
Department of Orthopedics, Mindong Hospital Affiliated to Fujian Medical University, Ningde Fujian, 355000, P.R.China.
Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Nov;27(11):1380-5.
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.
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.
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 lentiviral 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.
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.
构建猪骨形态发生蛋白2（BMP-2）基因重组慢病毒载体，检测BMP-2基因活性及骨髓间充质干细胞（BMSCs）成骨分化情况，为骨软骨组织工程的进一步研究奠定基础。
从2月龄巴马小型猪（体重15 kg）骨髓中分离BMSCs，取第2代BMSCs进行实验。采用重组DNA技术构建猪BMP-2基因慢病毒载体，以感染复数（MOI）为10、25、50、100和200转染BMSCs，通过荧光显微镜和倒置相差显微镜确定MOI的最佳值。用BMP-2重组慢病毒载体转染的BMSCs作为实验组（BMP-2载体组）；用空载体转染的BMSCs（空载体组）和未转染的BMSCs（未转染组）作为对照组。采用RT-PCR、免疫组织化学染色和Western blot检测BMP-2 mRNA和蛋白的表达。然后通过碱性磷酸酶（ALP）染色、ALP活性和茜素红染色检测BMSCs的成骨情况。
成功构建猪BMP-2基因重组慢病毒载体，并经RT-PCR和基因测序鉴定，BMP-2重组慢病毒载体成功转染BMSCs。在转染的BMSCs中可见绿色荧光蛋白，尤其是在MOI为100时表达最佳。免疫组织化学染色和Western blot显示，BMP-2重组慢病毒载体转染的BMSCs能持续稳定地高水平表达BMP-2蛋白。培养2周后，观察ALP的表达和钙结节的形成情况。
成功构建猪BMP-2基因慢病毒载体并转染至BMSCs，其能持续稳定地高水平表达BMP-2基因和蛋白，并诱导BMSCs分化为成骨细胞。
