| ObjectiveTo Investigative the influence of TGF-β2on proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells,and to explore the most suitable concentration for the proliferation and osteogenic differentiation within the concentration of TGF-β2ranging from0.1to10ng/ml in cells of the bone marrow mesenchymal stem.The aim is to provide reliable theoretical basis for further laboratory studies and in vivo studies.Method1. Taking a New Zealand white rabbit,remove bilateral femoral shaft, get bone marrow mesenchymal stem cells.Placed it in an incubator,3-4days to observe and medium was changed to cells covered passage, this time denoted by PI generations; When spread P3generation using immunohistochemistry adherent cell surface markers CD44, the resulting cells can be confirmed by bone marrow mesenchymal stem cells.With P1, P3, P5three generations of cell lines by MTT cell growth curve, comparing three generations of cell growth and viability, optimal cell lines selected for subsequent experiments.2. The P3generati re set three wells to detect alkaline phosphatase activity,the aim is to determine different concentrations of TGF-β2on bone marrow mesenchymal stem cells into osteoblast differentiation capacity;Each concentration were also set up three holes to make MTB detection of free calcium concentration to study whether TGF-β2promoted bone marrow mesenchymal stem cells into bone induction, and finded the best concentration of TGF-β2activity.3. The three generation of bone marrow mesenchymal stem cells were inoculated in the culture bottle, respectively, on the first day, three days, five days, seven days to remove the four time points after induction cells, discard the medium, washed once with PBS; then2ml4%paraformaldehyde fixed cells in30minutes; PBS cells were washed twice, each hole by adding lml Sin red staining working solution, incubated at room temperature for3-5minutes;washed3times with PBS, calcium accumulation was observed under a microscope.Qian red staining,calcium accumulation was observed under a microscope.Result1. After bone marrow mesenchymal stem cells isolated and cultured hematopoietic cells initially incubator ingredients more with time, these impurities or with cell necrosis was changed slowly removed in future be able to see part2adherent cells begin to grow about4days shows a large number of adherent cells, as colonies, cells begin to fusiform, resembling fibroblasts, radial growth. Cultured to12days, more than90%of cell fusion to be passaged culture to the third generation after bone marrow mesenchymal stem cells on the more pure, very few impurities cells. Cultured cells were analyzed by immunohistochemistry and found that bone marrow mesenchymal stem cell-specific antibody CD44(+), CD35(-). Between cultured cells proved to bone marrow mesenchymal stem cells.2. MTT detect cell growth kinetics, the experimental results showed that:bone marrow mesenchymal stem cells in a medium containing TGF-β2in growth absorbance absorbance values than that of the control group (no TGF-P2medium) in the light absorbance value is higher. Group B, C, D, OD values at different time points were higher than in group A, namely B, C, D groups were promoting bone marrow mesenchymal stem cell proliferation; Group C OD values at different points in time high in group A, B and D, indicating the group C compared with the other groups proliferation strong, that is, when the medium of TGF-P2concentration1.0ngl/ml, can get the most aggressive cell growth and proliferation.3.Alkaline phosphatase determination to promote bone marrow mesenchymal stem cells into osteoblasts optimal concentration of active TGF-β2, the experimental results show that:A group (no TGF-β2) in alkaline phosphatase has been at a low level of expression, and ALP expression in the rest of the group was significantly higher than its further line pairwise comparison, found B, C D group Youyi group C (TGF-|32concentration of1.0ng/ml) the expression of alkaline phosphatase activity the highest Description Group C (TGF-β2concentration1.0ngl/ml) to promote bone marrow mesenchymal stem cells into osteoblasts strongest effect.4. Methyl thymol blue (MTB) method for the determination of free calcium, applied methyl thymol blue (MTB) method for the determination of free calcium, B, C, D group than its calcium concentration in the control group (no TGF-β2culture y1) high, indicating that B, C, D groups were promoting bone marrow mesenchymal stem cells osteogenic effect; group C in the calcium concentration at different time points higher than in group A, B and D, indicating that compared with group C other groups to promote bone marrow mesenchymal stem cells osteogenic effect is strong, that is, when the medium TGF-β2concentration1.Ongl/ml, you can get the most positive osteogenic.5. Qian red staining, can be observed under the microscope to observe calcium accumulation, and when the medium at a concentration of TGF-β21.0ngl/ml, maximum calcium nodules.Conclusion1. between trypan blue staining of bone marrow mesenchymal stem cells, cell viability was confirmed by TGF-β2no damage to cells.2. TGF-β2on rabbit bone marrow mesenchymal stem cells promote the proliferation and optimal concentration is about1ng/mL;3. TGF-β2may promote bone marrow mesenchymal stem cells express ALP, and the optimal concentration of about1ng/mL;4. TGF-β2can improve bone marrow mesenchymal stem cells into bone induction, and the optimal concentration of about lng/mL. |
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