Study On The Proliferation And Differentiation Of Bone Marrow Mesenchymal Stem Cells Induced By Cerebrolysin, Fasudil

Objective:To study the method to maintain stem cell characteristics - self-renewal, and multi-directional differentiation potential in its isolation, culture and amplification for rat bone marrow mesenchymal stem cells (BMSCs), and to investigate the possibility and the proper condition that BMSCs could be proliferated and differentiated into neuron neuron-like cells by cerebrolysin,fsudil. To provide a theoretical and experimental basis for the BMSCs therapy for nervous system injuries and neurodegenerative diseases.Method:BMSCs were separated from rats bone marrow through femoral and tibial by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation.To investigate the optimal serumlevels and growth curve by MTT Assay.To investigate growth cycle and cell surface antigen CD34, CD44, CD29, CD90, CD45 and CD31 expression of BMSCs by flow cytometry.In vitro the BMSC were preindueed by bFGF and then induced by cerebrolysin and fasudil.The morphological changes were observed under phase contrast microscope, the specific markers of induced cells were identified by immunocytochemically with Nestin, NF, NSE, GFAP.Results:1.Isolation, culture and identification of BMSCs(1)Pure BMSCs were successfully obtained by adherence sieving from rat bone. After culturing primary BMSCs for 3 days, BMSCs were characterized by spindle-shaped spperearence and proliferated rapidly for 7 days, the cells were cluster, colony-like growth. Cells after 3 passage still on spindle.(2) 10%FBS of growth BMSCs is optimal serum levels.(3)Growth curve of passage cells showed that: the first 1-2d is latency, the first 3-4d is the logarithmic growth phase,6d after the cells enter the plateau phase.(4) Cell growth cycle analysis:cells in G0/G1 phase of 93.99%, S phase cells were 5.46%, G2/M0 0.54% of the cells. G0/G1 phase proportion of the total cell population reached 93.99%, while suggested that most of cells are not in period of proliferation. (5)Cell surface markers by flow cytometry:BMSCs expressed CD90, CD29, CD44. In contrast, no expression of the hematopoietic lineage marker CD34, CD45, CD31.2. Morphological changes after BMSCs induced(1)proliferation and differentiation of BMSCs induced by cerebrolysinlOng/mlbFGF pre-induction of 24h, the cells did not change. Then after exposed to cerebrolysin, the cell growth state was better than before and number of cells increased, and was statistically significant (P＜0.05). However, no obvious morphological changes, cell shrinkage, triangle-like cells appeared, but no obvious cell processes.(2) Proliferation and differentiation of BMSCs induced by fasudilFasudil induced group after 6h induced morphological changes gradually emerged bipolar shape, multipolar shape and conical cells present neuron-like cells.24h after induction, showing that more neuron-like cells, synapse formation and the gradual increase in cell processes are intercrossed present network-like connection.48h after induction, cell synapse fracture dissolution, the network-like connections disappear, the number of cells decreased.3.Immunocytochemical stainingThe control group showed no positive staining of the four antibodies. cerebrolysin group immunohistochemistry shows that a small number of Nestin, NSE, NF, GFAP positive cells, compared with the control group (P> 0.05).Fasudil induced experimental groups:6h after induction of Nestin, NSE, NF, GFAP antibody staining was positive (P<0.05).24h after induction the positiverate of Nestin, NF, GFAP were higher than 6h (P＜0.05); NSE-positive rate was lower than 6h (P> 0.05).48h after induction, Nestin, NF, GFAP-positive rate is higher than 24h (P<0.05); but the positive rate of NSE was in contrast (P> 0.05).Nestin, NF and GFAP:With the prolongation of induction time positive rate increased gradually (P<0.01).NSE:The positive rate of the 24h group was lower than those of the 6h and the 48h group, no significant difference(P> 0.05). Conclusion:High purity BMSCs were successfully obtained by screening adherence in vitro.After induction by cerebrolysin, no obvious morphological changes were found. It was after induction by fasudil that neuron like cells were induced to differentiate from BMSCs in vitro. By immunohistochemical analysis, these induced cells expressed neural cell-specific markers such as Nestin, NSE, NF, GFAP. It suggested that BMSCs might be used as nervel seed cells for stem cell therapy for brain injury and neurodegenerative diseases.