Construction Of Recombinant PIRES2-EGFP-hBMP₂ Vector And Its Effects On Human Gingival Fibroblast Phenotype

Periodontal regeneration is expected to reconstitute the injured tissues of the periodontal attachment apparatus physiologically and functionally, including gingiva, periodontal ligament, cementum and alveolar bone. Traditional periodontal therapy and guided tissue regeneration failed to fullfil the goal. Tissue engineering has been introduced into the field of periodontology, which contribute a new vista to periodontal regeneration. But there are some difficulties in finding proper seeded cells. Owing to its heterogeneity, periodontal ligament (PDL) cell is reagrded as the main source of cell to regenerate periodontal tissues and expected to be the seeded cells in previous studies, because of their potential of differentiation to fibroblasts, osteoblasts, and cementoblasts. However, there are also some disadvantages for PDL cell to be seeded cells, for example , extraction must be carried out to obtain PDL cell, which is often refused by patients in clinic.Gingival fibroblast is also the constituent of periodontal tissues, and easier to obtain clinically than PDL cell. gingival fibroblast have strong proliferation potential and even show some characters of osteoblasts when they are induced by some bone-inducing factors such as human bone morphogenetic protein-2 (hBMP2). hBMP2 is a kind of strong bone-inducing factor and plays a crucial role in periodontal regeneration. So, combining gingival fibroblast with hBMP2 may be used in periodontal tissue engineering to fulfill the periodontal therapy goal.This study tries to reform gingival fibroblast to be the seeded cells in periodontal tissue engineering by transfecting hBMP2 gene into gingival fibroblast. Several experiments as below are carried on in this dissertation:1. hBMP2 gene was successfully cloned from human osteoblastic osteosarcoma cell line OS-9901 by RT-PCR. After proofing by DNA sequencing, it was further constructed into a eucaryotic expression vector pIRES2-EGFP-hBMP2 gene via gene recombination technology, which paved the way to following investigation of hBMP2 on gingival fibroblast phenotype.2. hBMP2 gene was successfully transferred into human gingival fibroblasts by liposome transfection. After G418 screening, cell clones stably expressing hBMP2 were obtained. The hBMP2 expression was further confirmed by immunofluorescent microscopy, RT-PCR, Western blotting, and ELISA. These results showed that the transfected cells could stably express hBMP2 at least for nine generations.3,Observed the biological behaviours of gingival fibroblast transfected with hBMP2 gene stably, it was found that the shape of parts of gingival fibroblast changed from fusiform to polygonal; compared with parental cells, there were no obvious difference in doubling time and proliferative ability ; but enhanced both ALP activity and OC quantity of the hBMP2 gene transfected cells (P﹤0.01); the ALP synthesis was upregulated in the hBMP2 gene transfected cells by histochemistry staining, furthermore, mineral nodules developed in mineralization culture medium after gene transfection. Our results showed that the hBMP2 gene transfected cells expressed osteoblastic phenotype.To conclude, exogenous hBMP2 gene could stably express in gingival fibroblasts, and shift gingival fibroblasts to osteoblasts, and thus these gingival fibroblasts transfected with hBMP2 gene may serve as seeding cells for periodontal tissue engineering.