Vanadate Of Dexamethasone-induced Inhibition Of Rat Bone Marrow Mesenchymal Stem Cell Apoptosis Research

Objective: It is to utilize the all bone marrow adherence method to in vitro separate and culture rat BMSCs to provide stable and high purity stem cell.Method: Choose healthy male SD rat with 4– 6 years old and take out marrow of shinbone and thighbone under aseptic condition. The marrow adherence method could culture and separate mesenchymal stem cells and produce generation. Observe the 2nd generation cell in microscope for its shape and growth and produce growing curve. Then, induce it to be osteoblast cell and fat cell to make ALP, Alizarin Red, von kossa coloration, and oil red O coloration, while the flow cytometry is to test surface antigen label.Result: With the whole bone marrow culture method, the mesenchymal stem cells is found in microscope with shuttle shape or spindle shape and is seen with single shape after producing generation but maintaining high proliferation capability even until the second generation with S shape growing curve. The induced fat cell experiment group is found that the oil red O coloration is positive, osteoblast cell induced experiment group found the ALP, Alizarin Red, von kossa coloration, and oil red O coloration is positive but it in control is negative; and expression of CD44, CD90, CD105 is positive and it of CD19, CD34, CD45 is negative.Conclusion: The whole bone marrow culture method could obtain relative stable and reliable BMSCs. Under certain circumstances, it could be introduced to fat cell and osteoblast cell, while the surface antigen test complies with BMSCs. Objective: An in vitro SD rat BMSCs apoptosis model induced by dexamethasone was established to investigate the relationship between BMSCs apoptosis and the action time and the action concentration of dexamethasone. The inhibitory action and action mechanism of vanadate on cellular apoptosis induced by dexamethasone were also investigated.Method: Dexamethasone at 10-9M, 10-8M, 10-7M, 10-6M induced the second generation of BMSCs apoptosis at 3h, 6h, 12h, 24h respectively and the change of apoptosis rate was tested with flow cytometry. MTT Method was used to test the inhibitory action and best action concentration for dexamethasone induced BMSCs apoptosis by vanadate; TUNER Method and flow cytometry found that BMSCs apoptosis change upon dexamethasone action after 30 min. pretreatment of vanadate sodium. The RT-PCR tests the change of Caspase-3 and Bcl-2 after 30 min. of dexamethasone action on BMSCs. FAM-DEVD-FMK reagent could test the expression of factor caspases3, 8, and 9 after 30 min. pretreatment of vanadate sodium and dexamethasone acting on BMSCs.Result: TUNER Method and flow cytometry found that apoptosis cells increased after 12 hours of 10-8M, 10-7M, 10-6M of dexamethasone action, and increased with the prolongation of action time and increasing of action concentration. MTT method found that 10-7M vanadate pretreatment is helpful for protection over the dexamethasone induced BMSCs apoptosis. The flow cytometry had detected that 10-7M vanadate sodium pretreatment could greatly reduce the dexamethasone induced BMSCs apoptosis. This was also conformable to the test result of TUNER. RT-PCR found that the expression of Caspase-3 after dexamethasone action was increased compared to the blank control group, while Bcl-2 expression was decreased in this case. After pretreatment of 10-7M vanadate sodium, the expression of Caspase-3 was decreased compared with dexamethasone group, and the expression of Bcl-2 was increased compared with dexamethasone group. FAM-DEVD-FMK reagent could test the expression of caspases 3, 8 and 9 in dexamethasone treated BMSCs were all increased than blank control group, while in vanadate sodium pre-treated BMSCs were all decreased.Conclusion: We concluded that apoptosis induced by dexamethasone were related with action time and action concentration in a certain degree because dexamethasone induced BMSCs apoptosis by means of triggering extracellular pathway and mitochondria pathway. The vanadate salt had inhibitory action on the BMSCs apoptosis induced by dexamethasone, which might be realized by initiating the extracellular pathway and mitochondria pathway.