Experimental Study Of Transfecting EGFP Gene Into Osteoblasts Derived From Rabbit Marrow Stromal Cells

Objective To study the feasibility of transfecting EGFP gene into osteoblasts derived from rabbit marrow stromal cells, and to evaluate whether EGFP is an appropriate tracer of seeding cells.Methods Rabbit MSCs were aspirated from femur and directly planted in culture flask. After 24 hours, the medium was exchanged for the first time. Then the cells were expanded through subculturing in vitro. The passage four subcultured MSCs were cultivated in osteogenic medium. Ten days later, the activity of alkaline Phosphatase and the expression of collagen I were detected in the induced cells.pEGFP-C1 was transfected into induced osteoblasts with liposome and transient expression were evaluated by fluorescent microscope. 72 hours later, the cells were screened by osteogenic medium with G418. After G418 screening and cultured for 25 days, cell clones were observed with fluorescent microscope.Results The cells were adherent to the flasks and grew as spindle, polygonal or typically fibroblastic-like shape. The induced osteoblasts displayed no significant changes in morphology, but exhibited activity of alkaline phosphatases and expression of type I collagen. Osteoblasts transfected with EGFP gene expressed green fluorescent under fluorescent microscope.Conclusion EGFP could be expressed in osteoblasts induced by MSCs and used as a tracer of seeding cells for tissue engineering.