| Objective Malignant gliomas are the most prevalent type of primary brain tumors. Transplantation of progenitor cells is a promising approach for the treatment of gliomas. Bone marrow mesenchymal stem cells (BMSCs) are candidates for such a cell-based therapy, since they display an extensive tropism for gliomas in vitro and in vivo. However, few data are available on the factors involved in regulating BMSCs migration. The aim of our study was to investigate the migration behavior of BMSCs in neurogenic differentiation towards glioma and SDF-1α.Methods We studied BMSCs in neurogenic differentiation that migrate toward glioma and SDF-1αin three phases. (1) BMSCs were isolated by Percoll gradient centrifugation from rat, cultivation and amplification of BMSCs, and phenotypically identified by immunofluorescence staining. (2) BMSCs were induced to neural-like cells by the following steps: cells were treated with 10 ng/mL basic fibroblastic growth factor (bFGF) for 24 h, followed by 200μM butyl hydroxy anisole (BHA) plus 2% dimethylsulfoxide (DMSO) for 5 h. Finally, cells were maintained with DMEM (H) plus N2 for 48 h. We observed the morphology of BMSCs during neurogenic differentiation with inverted microscope. The expression of neural markers, Nestin,β-III-tubulin and NSE were analyzed by immunocytochemistry. (3) We investigated, by using the Dunn chamber, the directional migration of BMSCs in neurogenic differentiation towards the concentration gradients of chemoattractant. The migration pictures of differentiated cells were obtained by Leica AF6000, and analyzed by NIH Image J. 40 cells were selected randomly of each differentiation point for statistics to get migration speed, migration efficiency and migration trajectory of cells after induced 5 h. Next, we studied the chemotaxis and chemokinesis of BMSCs in neurogenic differentiation towards glioma by using the Boyden chamber. 10 fields of vision were selected randomly in the experimental group and control. Cells counted, the migration coefficient was calculated, migration coefficient = experimental group / control group.Results (1) We isolated and cultured rat bone marrow mesenchymal stem cells by using Percoll lymphocyte separation and discontinuous density gradient method. BMSCs can be passaged more than 20th generation. Immunofluorescence analysis showed that BMSCs were positive for CD29, CD71, CD90, CD106, and negative for CD34, CD45. (2) FGF, BHA and DMSO were used to induce the neural differentiation of BMSCs, after pre-induced 24 h, BMSCs became spindle. Treatment of BMSCs with BHA for 5 hours led to a dramatic change in morphology, such as cell body contraction and neural-like axis production. After maintained 48 h, cells took on more bifurcations and secondary bifurcations, while the control remained the morphology of BMSCs. The expression of neural markers, Nestin,β-III-tubulin and NSE were analyzed by immunocytochemistry. (3) Dunn chamber analysis revealed that both the migration speed and efficiency of cells induced by C6 conditioned medium and SDF-1αwere higher than control, demonstrating the chemotactic effect of both C6 conditioned medium and SDF-1αon BMSCs. These results were confimed by examination of migration tracks of individual cells. Moreover, BMSCs at different states of differentiation showed different degree of tropism for C6 conditioned medium and SDF-1α. The differentiated cells migrated significantly more than the control in Boyden chamber chemotaxis experiment. While there was no significant difference in chemokinesis experiment.Conclusion The directed migration of BMSCs was closely related to the differentiation states of these cells. The directed migration of pre-induced 24 h cells were the strongest. |
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