| Objective:To investigate the method of isolated and cultured rat bone marrow mesenchymal stem cells (MSCs) in vitro,and prove the purification of bone marrow mesenchymal stem cells co-cultured with glial cell line-derived neurotrophic factor (GDNF) can differentiate into neural cells.Method:The bone marrow cells were obtained from healthy SD rats, whole bone marrow primary culture, purified by repeated passage in vitro. The expressions of CD34, CD44, CD45, CD90cell factors were examined by flow cytometry, to identified whether it is the MSCs. MSCs were randomly divided in to control group and glial cell line-derived neurotrophic factor (GDNF) group. Cell morphology was continuously observed by inverted phase contrast microscope. Expression of nestin, neuron-specific enolase (NSE) were identified by immunocytochemical technique.Result:Cells appeared spindle-shaped and showed fibroblast cell-shape. Cells had high purity and good activity within forth generation. The positive rates of CD34, CD44, CD45, and CD90in MSCs were3.98%,98.74%,1.03%,98.08%respectively. Following induction, The cell body gradually shrinkage to the nuclear, deformation of cells gradually increased,MSCs presented dipolar, multipolar and pyramidal typical neuron morphology. In GDNF group, nestin was positive6hours after inducing and the positive result had peak value24hours after inducing. Neuron specific enolase (NSE) was positive12hours and had peak value48hours after inducing, the intensity of expression of above-mentioned indicators were gradually increased. But the induced cells apoptosis and fall off gradually after72hours.Maker expression of neurons and changes of morphology were not presented in control group.Conclusion:Isolating the bone marrow mesenchymal stem cells by the whole bone marrow culture method and finding that cells had high purity and good activity and could be induced to differentiate into neuron-like cells by GDNF. |
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