The Experimental Study Of DMOG Solution On The Effect Of Rabbit BMSCs Osteogenic Differentiation And Cell Proliferation In Vitro

purposeExplore the effect of proline hydroxylase inhibitor(DMOG) of rabbit femoral bone marrow mesenchymal stem cells to osteogenic differentiation and cell proliferation. for the specificity inducing bone marrow mesenchymal stem cells to osteogenesis differentiate provides experimental data and theoretical bases.methodschoosing a normal, healthy New Zealand white rabbit, the rabbits was put to death through the way of injecting pentobarbital,, remove the double femoral, behind put it in aseptic operation, and with DMEM/F12completely nutrient solution flush the medullary cavity, using the method of whole bone marrow culture, to remove impurities cells through adherent method, primitive cells have been subcultured three times,, using flow cytometry instrument to detect cell surface markers CD44, confirmed the cultured cells of bone marrow mesenchymal stem cells have high purity.The first step:using bone marrow mesenchymal stem cells for isolating, cultivating and testng;A in1d,3d,7d,12d, observing the growth situation of the original generation cells;B on the second and the fifth generation of cells, in1d,3d,5d,7d,9d,11d, to test the cell growth kinetics;C using cell immunochemical to detect the CD44expression of P3’cell surface;D observing the different concentrations of DMOG solution on the optical density value of the cell and ALP expression effect.The second step:We divided cells into three groups, A the group contains the osteogenetic differentiation culture solution and joining DMOG solution (500umol/L) B the group contains the osteogenetic differentiation culture solution C the group contains simple culture solution.A:in14days and21days, using Gomori calcium cobalt to observe BMSCs alkaline phosphatase expression and the influence of the calcium content of three groups.B. in5,10,15,20d, using MTB colorimetric method to detect intracellular calcium ions quantitative of three groups.C. in5,10,15,20d, using ELISA method to test the osteocalcin protein in cells,ResultAfter48hours, the cells was growing adherent growth visiblely, like fibroblasts, about96hours later, changing to long fusiform cells form, after purification for some times, the cells had putted into testing by immunochemical method, the results show cells bone that we have separated individual CD44(+);When the concentration of DMOG belongs to100umol/L-500umol/L, following DMOG solution concentration increased, the cultured cells OD value is declining, but the expression of alkaline phosphatase have rised continuously, using statistics to analysis data, comperd with others group DMOG solution, found the concentration of100umol/L-500umol/L has inhibitory effect on cell proliferation, but P>0.05, there was no statistically significant difference, to analyze the expression of ALP, P<0.05, the difference was statistically significant;When the concentration of DMOG belongs to500umol/L-2000umol/L, following DMOG solution concentration increased, the cultured cells OD value is declining, and the expression of alkaline phosphatase declining constantly, using statistics to analysis data, comperd with others groups,found that the concentration of500umol/L-2000umol/L has inhibitory effect on cell proliferation of DMOG solution, and P<0.05, the difference was statistically significant, to analyze the expression of ALP,P<0.05, the difference was statistically significant;Using Gomori cobalt staining to Detect alkaline phosphatase,showed that the calcium containing500umol/LDMOG nutrient solution can significantly increase the expression of ALP in osteogenesis cells differentiation, using Von-Kossa staining to detect calcium deposits improved,showed that culture medium containing500umol/LDMOG can obviously increase calcium deposits;Using MTB staining method to quantitative detection of calcium, concluded containing500umol/LDMOG can make the calcium content of the culture medium significantly increased, compared with the other two groups, P<0.05, the difference was statistically significant;Using ELISA method to detect the expression of osteocalcin in the cell protein quantitatively, concluded the concentration containing500umol/L DMOG can significantly increase the express of osteocalcin, compared with the other two groups, P<0.05, the difference was statistically significant;Conclusion1. The cells which we obtained from the experimeng is the bone marrow mesenchyal stem cells.2. The concentrations of containing DMOG500umol/L,It is not obvious inhibition on cell proliferation, and the expression of ALP is strongest;3. The nutrient solution Containing500umol/LDMOG can significantly increase the content of calcium and increase osteocalcin expression in the differentiation of BMSCs development.