| Objective As traditionary therapeutic method can't completely cure malignant gliomas, they are the most prevalent type of primary brain tumors. While Bone marrow mesenchymal stem cells (BMSCs) are candidates for such a cell-based therapy, since they display an extensive tropism for gliomas both in vitro and in vivo. However, few data are available on whether BMSCs differentiated have the same tropism or the mechanism that regulates BMSCs migration. The aim of this study is to investigate the migration behavior of BMSCs in neurogenic differentiation towards platelet-derived growth factor (PDGF), a factor secreted by malignant gliomas, and the mechanism involved in the migration.Methods We studied the effect of PDGF on BMSCs migration through four parts. The first three parts were about the migration of BMSCs in neurogenic differentiation towards PDGF, whereas the molecule mechanism of the migration was investigated in the last part. The concrete contents of each part are as follows: (1) BMSCs were isolated by Percoll gradient from rat bone. Then BMSCs were cultivated, amplificated and phenotypically identified by immunofluoresce staining and differentiation capability. (2) BMSCs were induced to differentiate into neural-like cells by the following steps: BMSCs were treated with 10 ng/ml basic fibroblastic growth factor (bFGF) for 24 h, followed by treated with 200μM butylated hydroxyanisole (BHA) plus 2% dimethylsulfoxide (DMSO) for 5 h. Finally, DMEM plus N2 was used to maintain the differentiation for 48 h. We observed the morphology of BMSCs during neurogenic differentiation with inverted microscope, and the expression of neural markers, nestin,β-III-tubulin and NSE were analyzed by immunocytochemistry. (3) We investigated, using the Dunn chamber, the directional migration of BMSCs in neurogenic differentiation towards the concentration gradients of PDGF. The migration pictures of differentiated cells were obtained by Leica AF6000, and analyzed by NIH Image J. Thirty cells were randomly selected from each state for statistical to get migration speed, migration efficiency and migration trajectory of individual cell. (4) Using time-lapse video, the effect of Rac1 on the BMSCs migration induced by PDGF were studied. While the effect of LY294002, the inhibitor of PI3K, on the BMSCs migration induced by PDGF were studied by Dunn chamber and time-lapse video. The migration pictures of every state cell were obtained by Leica AF6000, and analyzed by Image J. Thirty cells were randomly selected from each state for statistical to get migration speed and migration efficiency.Results (1) We isolated and cultured rat bone marrow mesenchymal stem cells using Percoll lymphocyte separation and discontinuous density gradient method. BMSCs can be passaged for more than 20 generations. Immunofluorescence analysis showed that the cells were positive for CD29, CD90, CD106, and negative for CD34. Besides, they can be induced to differentiate into osteocytes and adipocyte. Thus the cells have the basic characteristics of BMSCs. (2) BHA, bFGF 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 h led to dramatic changes in morphology, such as cell bodies contraction and neural-like axis production. Then maintained 48 h, cells took on more bifurcation and secondary bifurcation, while no changes were found in the control group. The expression of neural markers, nestin,β-III-tubulin and NSE were found in BMSCs in neurogenic differentiation by immunocytochemistry. (3) Dunn chamber analysis revealed that both the migration speed and efficiency of cells induced by PDGF were higher than control, demonstrating the chemotactic effect of PDGF on BMSCs. These results were confimed by examination of migration tracks of individual cells. BMSCs at different differentiation states showed different tropisms for PDGF: the migration speed of BMSCs undifferentiated, pre-induced 24 h and induced 5 h were significantly increased, while the migration efficiency of BMSCs undifferentiated, maintained 18 h and 48 h were increased notablely. (4) From the results of time-lapse video, we can see that the activation of Rac1 will enhance BMSCs migration induced by PDGF, on the contrary, down regulating Rac1 decreased the migration. The adding of LY294002 also depressed the migration speed and efficiency. Dunn chamber showed the same result. These repress that both Rac1 and PI3K play a role in BMSCs migration induced by PDGF.Conclusion BMSCs can migrate towards PDGF. Tropism of BMSCs for PDGF is closely related to their differentiation states. Both Rac1 and PI3K take part in this process. |
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