Experimental Study Of Rabbit Bone Marrow-derived Mesenchymal Stem Cells Cultured In Vitro Amplification Factors

Objective:To study rabbit bone marrow-derived mesenchymal stem cells cultured in vitro amplification of all conditions and the various factors affecting the comparison of the differences,and explore a simple,high efficiency of cell proliferation,stem cells used for wound healing and recycled tissues and organs to provide the necessary prerequisite for help.Method:1.From rabbits(age＜3 monthes)of intertrochanteric area under strict aseptic conditions vacuum extraction of bone marrow puncture 5-10 ml by density gradient centrifugation and paste the wall of separation or total separation and purification of bone marrow bone marrow-derived mesenchymal stem cells,37°C and 5%CO2 incubator eggs in fertility.Two methods of cultivating cells of the original colony of cloned cells,cell growth time and the number of centrifugal 16 d cells.2.Select the New Zealand white rabbits with different age groups under aseptic conditions in the inter-district femur bone marrow puncture vacuum extraction of 5-10 ml by density gradient centrifugation and paste the wall of separation and purification of mesenchymal stem cells,LG-DMEM(10%FBS)in a primary medium,and subcultured. Detect different age group of mesenchymal stem cells for cell colony formation and cell growth activity.3.Select the New Zealand white rabbits with age＜3 months,density gradient centrifugation and paste the separation wall in vitro bone marrow mesenchymal stem cells,LG-DMEM(10%FBS)culture medium. After the cells to be used LG-DMEM(10%FBS),LG-DMEM(15%FBS), LG-DMEM(20%FBS),HG-DMEM(10%FBS)suspended four medium-cells, Determination different media amplified the cultured cells,colony-forming efficiency,growth and cell morphology.4.Select good growth P4 generation osteoblast cell conditioned media induced changes in cell morphology were observed and Alizarin red staining.Results:1.By density gradient centrifugation and paste the wall of separation and purification of bone marrow-derived mesenchymal stem cells in 10%FBS,L-DMEM medium.In the control group,the same culture conditions separation from the whole culture of bone marrow mesenchymal stem cell proliferation time than the obvious density gradient centrifugation purification head.There are significant differences between the two groups(p＜0.05) 2.LG-DMEM(10%FBS),LG-DMEM(15%FBS),LG-DMEM(20%FBS), HG-DMEM(10%FBS)4 kind of medium,the LG-DMEM(15%FBS)group in cell growth amplification factor of 16.20±1.60 times,2-3 days to enter the logarithmic growth phase,the average cell colony formation rate is 6.11±1.17%.Comparison of LG-DMEM(10%FBS)group and the LG-DMEM(20% FBS)group in the cell growth cycle of amplification,the average rate of cell colony formation and cell growth activity,the difference is not significant.Cell growth with the increase of FBS concentration,morphology has changed. LG-DMEM(20%FBS)in the group in the late proliferative cells appeared in the process of the aging phenomenon obviously.HG-DMEM(10%FBS)in cell growth amplification multiple smaller than the other three groups,with an average of 11.40±2.07 times the average colony formation rate 2.67±1.00%, and cell proliferation rate lower than the other three groups.3.Different age groups in the bone marrow mesenchymal stem cells at different rates,age ＜3-month-old rabbit bone marrow mesenchymal stem cell proliferation best cell growth activity was significantly better than the other two groups.4. Mesenchymal stem cells induced osteoblast After 18 d,positive staining in selenium,calcium nodule formation.Conclusions:1.Under the same conditions density gradient centrifugation and adherent of cultured bone marrow-derived mesenchymal stem cells is better than the whole bone marrow culture,and cell proliferation good.2.LG-DMEM (15%FBS)than the other three medium medium better suited bone marrow-derived mesenchymal stem cells cultured in vitro amplification,and better maintain the normal cell morphology and biological activity.3.the rabbite with age＜3months contain more bone marrow mesenchymal stem cells,and the proliferation of biological activity was superior to the ageing group.