The Expression Of Osteoprotegerin And Biological Characteristics Of The Rat Bone Marrow Stromal Cells Transfected By Osteoprotegerin Gene

Objective To investigate the osteoprotegerin(OPG)gene expression and analyzed the biological characteristics of BMSCs after transfected with plRES2-EGFP-OPG in vitro.Methods The BMSCs were isolated from bone marrow in SD rats about four-week-old. BMSCs were divided into three groups: plasmid group,blank plasmid group and non-transfection group, the cells in the plasmid group were transfected with plRES2-EGFP-OPG, while cells in the blank plasmid group were transfected with plRES2-EGFP and cells in the non-transfection group received no special treatment. At 48 hours after transfected, EGFP were detected under laser scanning confocal microscope; the expressions of mRNA of OPG gene in BMSCs was analyzed by RT-PCR; OPG protein in transfected cells were determined by immunocytochemistry; the proliferativity cells were assayed by methabenzthiazuron(MTT) method; the ability of transfected cells to osteoblast differentiation through alkaline phosphatase.Results 1. The morphological characteristics of BMSCs in primary and passage culture: the bone marrow cells of the initial separation were round; after 24 h, a small amount of cell adhesion; after 96 h, the growth of adherent cells were mainly spindle fibroblast-like cells; the cells of clones were formed after sub-cultured bottle; after passage, adherent cells grow and divide phase increases, and cells continue proliferation differentiation into a form like homogeneous spindle.2. The laser scanning confocal microscope showed that: the group of transfected plasmid expressed green fluorescent protein, no expression of untransfected group. 3. The expressions of OPGmRNA and protein by RT-PCR and immunohistochemical analysis: the expressions of OPGmRNA were observed in plasmid vector group, there was no expression of empty vector transfected group and untransfected group. The OPG protein was found in plasmid vector group; empty vector transfected group and not as a negative.4. MTT method assess BMSCs growth and proliferation: after transfected, cells grew well and spread extensively and proliferated rapidly on AFDPB. There was no significant difference in the cell number between plasmid vector group, empty vector transfected group and untransfected group (P >0.05).5. Alkaline phosphatase (ALP) staining: the ability of synthesis ALP was significantly increased after BMSCs transfected plRES2-EGFP-OPG, the blue-positive granules was found in cytoplasm, the expression of ALP was significantly higher than empty vector transfected group and not(P < 0.05).Conclusion 1. Successfully isolated and cultured BMSCs, and furter proofs BMSCs can be proved as an ideal seed cells to gene bone enhanced tissue engineering.2. BMSCs can stable and efficient expression of OPG after transfected OPG gene, and established OPG gene-modified's bone marrow stromal cells in transient gene expression system.3. After transfected plRES2-EGFP-OPG, BMSCs proliferation is not affected and have some biological function, can promote their differentiation into osteoblasts.