Experimental Study Of Bone Induction By Muscle-derived Stem Cells Transfected By Recombinant BMP2 And Angiopoietin-1 Adenovirus

Bone defects and fracture non-unions are the clinical probles at present. The results of repair of bone defects by studies of bone engineering are still not all satisfied.The perfect seed cells and vectors for delivering osteoinductive growth factors remained to be found. The muscle-derived stem cells( MDSCs) isolated from rodents skeletal muscle, which posses the characteristics of extensive origin, and rapid amplification in vitro and osteoinductivity, may prestent the seed cells of bone engineering. Bone morphogenetic protein-2(BMP2) is a potent osteoinductive factor, while vascular endothelial growth factor(VEGF) plays a role in BMP2 induced bone formation, suggesting the probable synergistic effects of osteoinductive growth factors and angiogenic factors. Angiopoietin-1(Ang1) is a newly characterized angiogenic factor , and its roles in bone repair are to be elucidated. Therefore ,the present study are conducted to induce bone formation by MDSCs transfected by recombinant adenovirus encoding BMP2 and/or Ang1 . Six parts of the present study are included:The study of part one addressed the expression of Ang1 and its receptor Tie-2 after fractures of radius in rabbits. As shown by immunostaining, the expression of Ang1 and Tie-2 receptor were detected in the osteoblasts,and osteoblastic progenitor cells at the fracture ends, and in the endothelial cells and pericytes of new vessles ,as well as in karyotes in the bone morrow cavity. The results of RT-PCR and ELISA demonstrated that the expression of Ang1 and Tie-2 could be detected by 8 hours after fracture. The expression of Ang1 and Tie-2 receptor reached its peak value by 72 hours and one week respectively after fractrue, and reduced gradually afterwards. The expression of Ang1 and Tie-2 returned to base-line 8 weeks after fracture. The expression of Ang1 and Tie-2 coincided with the phase of organization of hematoma and primary callus formation in the course of fracture healing, suggesting that Ang1 and Tie-2 probably play a role in the vasculogenesis of fracture healing.The isolation, identification and induced differentiation of muscle-derived stem cells (MDSCs)of rabbit were included in part two. The primary MDSCs were isolated from skeletal muscle of rabbit by modulating the method of isolating MDSCs of mice, and the putative MDSCs were identified by immunostaining specific for Bcl-2 and desmin. The putative MDSCse were cultured in differentiation medium of osteogenic lineage, and the expression of alkaline phosphatase , type â… collagen and osteocalcin in the induced cells were detected by immunostaining 2 weeks after culturing,indicating that the induced cells differentiated into the osteoblastic lineage.The putative MDSCs were also cultured in differentiation medium of myogenic lineage for 2 weeks, and polynucleate cells or nuclei-chain formed in the cultured cells, indicating that the putative MDSCs could differentiate along the myogenic lineage. The results of this study demonstrated that osteoblastic progenitor cells presented in the skeletal muscle; MDSCs posseses the characteristics of multilineage differentiation, and it is a kind of tussue-specific stem cells.The MDSCs presents osteoinductive potential,and it could be used as seed cells of bone engineering and the target cells of gene therapy .In part three, recombinant adenovirus encoding human angiopoietin-1 was constructed.(AdAng1)and was used to transfect MDSCs in culture. The expression of Ang1 in the transfected MDSCs was shown by immunocytochemstry. The total RNA and total protein of the transfected MDSCs was extracted ,and the quantity of Ang1 expression was assessed by RT-PCR and ELISA. The results demonstrated that AdAng1 could effectively express the target protein Ang1in the transfected MDSCs, and immunocytochemical staining showed the positive particles of Ang1 in the transfected MDSCs. The results of RT-PCR and ELISA analyses demonstrated that the expression of AdAng1 in the transfected MDSCs reached its peak value by 9 days after transfection, and the time of effective...