Construction of seed cells with the stable expression of human bone morphogenetic protein 2 gene for bone tissue engineering / 中国组织工程研究

ABSTRACT

BACKGROUND: Because of the non-homology of protein and gene between human and animals, to promote osteogenesis or spinal fusion of animals by construction of tissue-engineered bone with the human gene has influenced the experimental validation.OBJECTIVE: To construct the seed cell line for bone tissue engineering with stable expression of human bone morphogenetic protein 2 (hBMP2).METHODS: The full-length hBMP2 gene was cloned from human muscle tissues by nested RT-PCR and transfected to human bone marrow mesenchymal stem cells (hBMSCs) with lipidosome. The transfected hBMSCs were cultured with G418 in vitro to screen and purify the cells. A series of analyses such as RT-PCR, dot-ELISA, immunohistochemstry and alkaline phosphatase activity analysis were performed to evaluate the situation of hBMP2 expression and secretion at 48 hours and 3 weeks after the transduction. hBMSCs transduced with empty plasmid and the normal hBMSCs served as positive control and blank control groups, respectively, which were used for observation of cell growth, proliferation and biological characteristics of transfected cells. RESULTS AND CONCLUSION: The transfected hBMSCs appeared in small groups or clusters, and had a good proliferation after subculture in vitro. Some G418-resistance cell clones and calcium nodules were found when cultured with G418 in vitro. No significant difference was noted in the cell proliferation between the hBMP2 transfection group and two control groups. The ALP activity in the hBMP2 transfection group remained significantly higher than that in the two control groups (P < 0.01). At 48 hours and 3 weeks after transduction, hBMSCs could express actively hBMP2 by RT-PCR monitoring, and had a positive reaction of dot-ELISA and immunohistochemical analysis. The expression of hBMP2 gene in the experiment group at 48 hours was significantly higher than that at 3 weeks after transduction while there was no expression of hBMP2 gene in the two control groups. The above results show that the hBMSCs transfected by hBMP2 gene not only have potentials of normal proliferation and osteogenic differentiation, but also can stably express hBMP2.