| OBJECTIVE:To establish a method of isolation, purification and cultivation of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro, and provides cells basis for the further research.METHODS:Obtaining young rabbits of BMSCs, using the whole bone marrow adherence method to isolated, cultured, purified of BMSCs in vitro. Observed cell morphology and assess multi-directional differentiation capacity. The BMSCs cell of Adipogenic induction was identificated by oil red O staining. The BMSCs cell of Osteogenic induction was identificated by Alkaline phosphatase staining and alizarin red staining.RESULTS:The primary BMSCs proliferated as colony mode,were spindle shape, cell clusters arranged radially; the passage BMSCs were distributed uniformly with same morphology, growed and breeded rapidly, short subculturing period. The fifth generation of BMSCs growth curve like S-types, cultured for2days then enter the logarithmic phase, proliferated fastest in4days. The rabbits of BMSCs were positive to oil red O staining after adipogenic induction for14days, lots of round or oval-shaped lipid droplets in cytoplasm were observed under the microscope; cells were positive to alkaline phosphatase staining and alizarin bordeaux staining after osteogenic induction for21days, light-proof of increased density cell were aggregate could be observed by microscope.CONCLUSION:The whole bone marrow adherence method is a feasible way to isolated, cultured and purified BMSCs, which simple operation. The BMSCs with high purity, high viability, biological characters stability, has the potential ability of self-renewal, proliferation and multi-directional differentia-tion biological characters as the BMSCs. It's qutie an ideal method to culture bone marrow mesenchymal stem cells. BACKGROUND:Rhizoma drynariae can promoting fracture healing, preventing and treating osteoporosis, has great significance for bone rehabilita-tion and reconstruction. However, its specific mechanisms is remain unclear.OBJECTIVE:Observeing the influences of assemble flavone of rhizoma drynariae on proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs) and promote BMSCs differentiate to the osteogenesis, to discuss the possible mechanism of Rhizoma drynariae promoting fracture healing.METHODS:The rabbit BMSCs were isolated, proliferated and purified by whole bone marrow adherence method in vitro. The influence on the rabbit BMSCs proliferation caused by different concentration of assemble flavone of rhizoma drynariae was observed with methyl thiazdyl tetrazolium (MTT) assay; rabbit BMSCs were divided into experiment group, positive control group and blank control group cultured in vitro, then to observe the morphology by phase-contrast microscope and scanning electron microscope(SEM), comparison of the alkaline phosphatase activity at different times, osteogenesis identified by alkaline phosphatase staining, Immunohistochemical SABC method for I collagen protein, alizarin bordeaux staining and von Kossa staining; application of computer software calculated von Kossa staining calcification area.RESULTS:After co-culturing by different concentration of assemble flavone of rhizoma in vitro, the results of MTT showed that the assemble flavone of rhizoma drynariae with10-5?10-7mmol/L had promoted proliferation on rabbit BMSCs, especially the10-6mmol/L group. After10-6mmol/L assemble flavone of rhizoma drynariae induction, the cell clusters aggregated progressively with the character of light-proof under phase-contrast microscope, osteoblast-like cell morphology and calcium node formation could be observed under SEM; cells were positive expression of alkaline phosphatase and collagen-?, positive to alizarin bordeaux staining and von Kossa staining. The alkaline phosphatase activity and percentage of calcification area was more higher in experiment group and positive control group compared with that of blank control group with significant difference (P<0.05), however, experiment group was significantly lower than positive control group (P<0.05).CONCLUSION:A certain concentration of Assemble flavone of rhizoma drynariaecan can accelerate the rabbit BMSCs to proliferate, but effect of10-0mmol/L group was most obvious; and10-6mmol/L assemble flavone of rhizoma drynariae can enhance rabbit BMSCs osteogenesis, but the ability is inferior to the positive control group. |
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