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Objective: In recent years, studies have shown that bone marrow mesenchymal stem cells (Bone marrow stromal cells, BMSCs) can be induced in vitro to differentiate into nerve cells, BMSCs is expected to become the nerve cell transplantation seed cells to treat diseases of the nervous system, but BMSCs induced to differentiate low , differentiation and cell survival after a short time can not be effectively proliferation in order to meet the need of transplantation. How to be able to find a stable and efficient method of inducing and improve the survival of nerve cells after induction is one of the key issues that need to be resolved. Single sialic acid the four hexose ganglioside (monosialotetrahexosyl ganglioside, GM1) in the development of nerve cells, differentiation, have a very important role in the repair and signal transduction. Studies have shown that BMSCs induced to differentiate into neuron-like cells, GM1, we explore different concentrations of GM1 induce differentiation of BMSCs. Methods: isolated whole bone marrow of adult bone marrow, primary and subculture. Detected by flow cytometry BMSCs surface CD44, CD45, CD90, CD105 of expression to identify the purity. To containing 10μg/ml, 60μg/ml, 90μg/ml GM1 DMEM serum-free medium induced the fifth generation of BMSCs negative control group without any inducer. To 7d observed after induced changes in cell morphology and count, 6h after induction immunocytochemistry determination of cellular neural cell-specific surface markers neuron-specific enolase (NSE), neuron-specific microtubule-associated protein (MAP -2), to identify whether the induction of neuron-like cells and calculate the percentage of differentiation. Percentage differentiation among the groups comparison and statistical analysis. Determined by MTT assay 0.5h after induction, 6h, 24h, 3d cell growth conditions. Results: The cells were seeded given 2 days later washed with PBS, adherent cells were colony-like growth medium was changed visible. 8 to 10 days after the cells can be basically covered bottom, uniform spindle cells. A ratio of 1:3 passage after passage about 1 day cells can restore vitality and growth of adherent 5-6 days covered bottom. Passaged 4-5 generation cell morphology is relatively uniform, mixed small cell. Flow cytometry cell surface marker CD44 (), CD90 () CD105 (), CD45 (-), generally confirms the purity of BMSCs. BMSCs after pre-induction, the cells increase in size, shape longer. Inducing medium for 30 to 40 min, the cells begin to morphological changes in the cell body inward contraction, spherical or round to grow around protrusions, three-dimensional refraction enhanced; 4 h of induction, cell morphogenesis change in cell body further contraction in the formation of three-dimensional enhancement of processes, cell body, halos around and see a lot of bipolar and multipolar cells; 6 h after most of the cells to form a tapered, triangular, irregular shape, projections number of variable-length, cell between the protrusions are connected to each other, staggered into a network, showed typical nerve cell-like morphology. 3 days after the induction of neuron-like cells did not increase significantly. 5 days, the death of a small amount of the cell suspension, the projections extend reticulation; 7 day cell morphology with substantially the same 5 days more, but the suspension of dead cells. Negative control cells continued to show a uniform long fusiform cells, cell growth slowed significantly. Cells 6 h immunocytochemistry to detect different concentrations of GM1 group and negative control cells were MAP-2, NSE expression, but did not detect the expression of GFAP. 10μg/ml, 60μg/ml, 90μg/ml GM1 group NSE, MAP-2 cells positive rate than the negative control group was significantly higher (P lt; 0.05) Between the two groups: 60μg/ml, 90μg/ml group NSE, MAP-2 cells positive rate than 10μg/ml group (P lt; 0.05), 60μg/ml, 90μg/ml group of cells positive rate difference was not significant (P gt; 0.05). MTT assay showed that the neuron-like cells induced by GM1 better than the negative control group growth state. Conclusion 1, different concentrations of GM1 with BMSCs induced to differentiate into neural cells, induced BMSCs differentiation into nerve cells after 6 h, the highest rate. 2, medium and high concentration of GM1 induce BMSCs to differentiate into neuron-like cells, a higher percentage of the neuron-like cells induced growth state.
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