Influence Of The Rat Brain Homogenate On Bone Marrow Mesenchymal Stem Cells Differentiate Into Neuron-like Cells

Object: Bone marrow mesenchymal stem cells (BMSCs) have the potential of differentiating into neural cells. The experimental studies the characteristics of the bone marrow mesenchymal stem cells isolation, and cultivates high purity of mesenchymal stem cells in vitro. To investigate the influence of rat brain homogenate supernatant on bone marrow mesenchymal stem cells differentiate into neuron-like cells. To join in normal and damaged brain homogenate supernatant in inducing fluid, observe the induction rate and neurons vitality after induction in different groups.Method: BMSCs were collected from Wistar rats using whole bone marrow culture methods, cultured and passaged by adherent methods in vitro .The growth of BMSCs was evaluated by drawing cell growth curve. The expression of CD29, CD44 and CD45 on the surface of the fourth BMSCs was detected by flow cytometry. The 4th generation BMSCs were collected and inoculated in 6-well plates and 96-well plates by 105/ml concentration. BMSCs were pre-induced for 24 hours in DMEM medium containing 1mmol / L BME and 20% serum, then joined bFGF 20ng/ml, bFGF 20ng/ml + normal cerebral homogenate supernatant 100ul/ml, bFGF 20ng/ml + damaged brain homogenate supernatant 100ul/ml in medium respectively, induced 48 hours to cell differentiation. There was a control group without any inducer. Cell culture and induced process were observed their morphology change under inverted microscope. The expression of neurons specificity enzyme (NSE), microtubules related proteins - 2 (MAP - 2) and glial fibers acid protein (GFAP) expression on the surface of cells after differentiation 48 hours were observed by immunohistochemical method, and calculated the induction rate. To detected cells vitality after induction 24h, 48h, 3d, 5d and 7d by MTT method.Results: We observed that some adherent cells were polygonal cultivated 24 hours by inverted microscope .The adherent cells increased and proliferation after 72 hours and 70%～80% confluence was occurred in 2 weeks when subcultured . BMSCs grew and adhered after subculture 24 hours, bespread the cultivate bottle bottom in 5～7 days. During the following change fluid and subculture, the fusiform cells gradually performanced vortex shape and chrysanthemum shape. The 3th, 5th and 7th generation BMSCs were selected to be investigated their capability of proliferation curve. The curve showed there were no significant different characteristics among the three generations. BMSCs grew slowly until the third day, and then entered exponential growth phases. This period lasted until the 8th day, and then cells grew in platform period. Cell multiplication capacity decreased with increased subculture. Flow cytometric detected surface marker of 4th generation, the results for CD29, CD44 and CD34 expression rate was 99.8 percent, 99.7 percent and 2.2 percent. A few cells shrank to the nucleus direction after pre-induced 24 hours. These changes increased after joining inducer. The cells often showed the typical bipolar, multipolar and pyramidal-shaped structure, the control group cells grew without morphological changes. The induction groups of cells were NSE and MAP - 2 positive, GFAP negative by immunohistochemical. The control group was not express NSE, MAP-2 and GFAP. At 48 hours, the NSE expression rates of these cells in the bFGF group, bFGF+ normal cerebral homogenate supernatant group, bFGF+ damaged cerebral homogenate supernatant group, and control group were 44.50%, 63.10%, 73.30%, and 0, respectively. The MAP-2 expression rates were 45.70%, 65.30%, 75.60% and 0 .The immunohistochemical results showed the cells were positive in the inducer group, which suggested there were neuron-like cells growing. The MTT results showed that the cell vitality joining brain homogenate were better than that of control group, the damaged homogenate had a stronger function.Conclusion: The bone marrow adherent culture can develop a high purity of bone marrow mesenchymal stem cells. Rat brain homogenate supernatants improved the induced differentiation rate of BMSCs into neural cells and increased the vitality of cells, compared to normal brain tissue, the role of damaged tissue was stronger.