Study On The In-vitro Osteogenic Ability Of Rabbit Bone Marrow Stromal Cells Treated With Liposome-mediated Double Genes Transfection

Objective:(1)To explore the method of separating and culturing rabbit bone marrow stromal cells in vitro as seed cells for tissue engineered bone.(2)To explore the method of liposome-mediated double genes co-transfection of rabbit BMSCs(RBMSCs)with BMP-2 and VEGF-165 and to clarify the ability of cell proliferation of double genes co-transfection of rabbit BMSCs.(3)To observe the interaction of BMP-2 and VEGF-165 genes during expression in RBMSCs and to identify the osteogenic ability of RBMSCs co-transfected with BMP-2 and VEGF-165.Methods:(1)Bone marrow was extracted from healthy New Zealand rabbits and separated using density gradient centrifugation,followed by proliferation of RBMSCs and identification.(2)RBMSCs in Group A,B,C and D were treated using Lipofect-amine2000 liposome and transfected with p IRES-h BMP2-h VEGF165,p IRES-h BMP2,p IRES-h VEGF165,and blank plasmid,RBMSCs transfected were observed using fluorescence microscope and MTT growth curve was prepared(3)the expression of BMP-2 and VEGF-165 were determined with morphological observation and enzyme linked immunosorbent assay(ELISA).The osteogenic ability was also determined by measuring alkaline phosphatase,bone collagen ?,and osteocalcin.Results:(1)The morphology change of RBMSCs:The single round nucleated cells are observed in culture-medium at the beginning of separating.The increase of Anchoring cells incresed at 48 h after separating,and the morphology of cells turn into triangle and commashape..After 6-10 days of culture,volume of many adherent cell increases gradually,colony formation collected and increasing cell morphology became elongate and polygonal,contains 1 or 2nucleoli;confluent monolayers of cells appeared at 14 days of cultured,most of the cell nuclei disappeared.RBMSCs cells were spherical or elliptic after passage and quickly return to shuttle shape.Cell shape became spindle after completely adherent and cell density increased.The proliferation rate of cells was faster After passage.After 5 to 7 days,the cells covered the culture bottle,and the cells were uniformly distributed in a spiral shape.No obvious morphological changes were observed after continuous passage.RBMScs after gene transfection was in shuttle or polygon shape,the speed of cells proliferation was decreased than before transfection.After 24 hours of transfection,a small amount of green fluorescence was observed in the transfected group,which was proved to be successful.After transfection for48-72 hours,the fluorescence gradually increased,and the fluorescence intensity was strong 4d after transfection.(2)Based on the MTT results and observation results of fluorescent light after transfection,the OD value increased with time increased.The increased OD value of each group has no statistically significant.OD value of P3 normal RBMSCs had no obvious change in 1 and 2 d,OD value showed a clear increase at 3d,and reach the top at 7d.the OD value increased of A group had no obvious change in 1 d,value gradually increased at 2d,the growth rate is less than the normal RBMSCs cells,OD in the peak at 6D and then decreased slowly into the platform.B group and C group OD value and growth curve of cells was same as A group,had no s statistically significance.The cells of D group and P3 normal rabbit BMSCs cells had no obvious statistical significance compared to OD changes.(3)After 4d of transfection,satisfactory expression of BMP-2 and VEGF-165 were measured with ELISA in RBMSCs transfected with p IRES-h BMP2-h VEGF165.The expression of BMP-2 and VEGF-165 was significantly higher compared with RBMSCs transfected with p IRES-h BMP2 or p IRES-h VEGF165(P<0.05).No expression was observed in RBMSCs treated with blank plasmid.(4)After 4d of transfection with plasmid of optimal concentration,expressions of Type I collagen,alkaline phosphatase(ALP),and osteocalcin in p IRES-h BMP2-h VEGF165-transfected group were significantly higher than RBMSCs transfected with p IRES-h BMP2 or p IRES-h VEGF165,with significant osteoblast morphology and osteogenic activity observed.Conclusion:(1)Based on the MTT results and observation results of fluorescent light after transfection,the ability of cell proliferation of RBMSCs co-transfected with BMP-2 and VEGF-165 is same as RBMSCs solely co-transfected with BMP-2/VEGF-165,but little lower than normal RBMSCs.The result has no statistical significance.(2)BMP-2 provides obvious osteogenesis promoting effect,which could promoted mutually with VEGF-165.(3)RBMSCs co-transfected with BMP-2 and VEGF-165 provides better osteogenic activity compared with single gene-transfected RBMSCs or untreated RBMSCs,which provided further evidence for long bone defect repair with tissue engineered bone constructed using compound artificial bone allograft...