Construction of pIRES2-EGFP-hBMP2 and its effect on the phenotype of gingival fibroblasts
TANG Hao-Zhe1, WANG Qin-Tao1, DONG Guang-Ying1, MA Zhi-Wei1, DING Ao1, LI Chang-Chun2, SUN Ying-Chun2, WANG Li31College of Stomatology, 3Faculty of Preclinical Medicine, Fourth Military Medical University, Xi'an 710033, China, 2Department of Maxillofacial Surgery, Tanggu Hospital of Stomatology, Tianjing 300450, China
AIM: To construct and identify the eukaryotic expression vector pIRES2-EGFP-hBMP2 which expressing human hBMP2 and to observe its effects on the phenotype of human gingival fibroblast. METHODS: hMBP2 gene was cloned from human osteogenic sarcoma by RT-PCR and inserted into T-vector. After DNA sequencing verification, it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP .After identification by double digestion, the pIRES2-EGFP-hBMP2 was transfected into HEK293 cells to verify the expression by fluorescence microscope and Western Blot. The construct was transfected into human gingival fibroblast to detect the effects of hBMP2 on the proliferation, ALP activity, content of OC and the ex vivo mineralization capacity of fibroblast by MTT. RESULTS: Human BMP2 gene was cloned successfully and verified by double digestion and DNA sequencing.The constructed eukaryotic expression vector pIRES2-EGFP-hBMP2 expressed hBMP2 correctly. hBMP2 did not affect the proliferation of human gingival fibroblast but increased the ALP activity, OC content and ex vivo mineralization capacity. CONCLUSION: The eukaryotic expression vector pIRES2-EGFP-hBMP2 expressing hBMP2 is constructed successfully and this gene promotes human gingival fibroblast to differentiate toward osteoblast.