| Objective:Neural stem cells (NSCs) is a stem cell that has the potential to differentiate into neurons, astrocytes and oligodendrocytes and self-renew sufficiently. These cells provide a new way for therapying neurodegenerative disease and injured brain in clinic. However, whether in vitro or in vivo findings of the study, the proportion differentiating into neurons from NSCs is lower than that of glial cells. It is difficult to achieve alternative for injury and disease causes the loss of neurons purpose.Bone marrow stromal cells (BMSCs) derives from marrow multipotential stem cells and can express many cytokines and growth factors, and these factors support the differentiation and maturation of hematolymphopoietic stem cells. Recent studies suggected that many cytokines produced by BMSCs are able to regulate NSC differentiation, proliferation and survival, including brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin and so on. Our laboratory confirmed that BMSCs could directly induce the differentiation of mesencephalic NSCs into high proportional neurons. The soluble elements that BMSCs secreted to the culture medium of played an important role in this process. Furthermore, it remains unclear what is these soluble molecules and how these cytokines play those roles.Mitogen-activated protein kinase (MAPK) commonly found in a variety of biological cells, and transduces extracellular signal into a cellular response. Thereby MAPK pathways regulate cell growth, differentiation and apoptosis process. This study aimed at investigating the effect MAPK pathways on differentiation of NSCs. NSCs were cultivated by Neurobasal medium made of BMSCs conditioned medium (Neurobasal -conditioned Medium, N-CM). Three MAPKs signal transduction inhibitors were added into N-CM in order to observe the neural and glial differentiation of NSCs. The purpose of the study is making clear that the signal transduction mechanism of BMSCs regulating differentiation of NSCs and the impact of MAPKs signal transduction pathway in the role of this process.Methods:BMSCs were collected from femur and tibia of SD rats by flushing the bone marrow cavity the shaft, cellular suspension was centrifuged and planted into 75 ml culture of the bottle. When the 6 generations of BMSCs covered 85 percent of the bottle 85%, the culture medium was replaced by Neurobasal medium 6 ml. After 24 h, the centrifugal supernatant was collected as N-CM. The midbrain of newborn SD rats was removed from the brain, and mechanically dissociated into single cells. Tissue suspension was centrifuged, the tissue pallets were resuspended with proliferation medium (DMEM/F12 (1:1) adding the addition of 2% B27 and 20 ng / ml bFGF). After 7 days, the single NSC could proliferat to form spheres (NSCs spheres). Then, NSCs were passaged 5-7 days each. The second-generation or third-generation NSCs spheres were planted in the 35 mm pre-coated poly-L-lysine dishes. The culture medium was replaced into the medium adding signal transduction pathway inhibitor when cells adherented on the dishes. There were three groups in our study: (1) natural differentiation group: NSCs cultivated in simple Neurobasal medium; (2) control group: NSCs cultured in N-CM; (3) inhibitor group: NSCs cultivated in N-CM adding three inhibitors, i.e. 5μM PD98059, 4μM SB203580, 4μM Genistin to N-CM respectively. Immunocytochemistry was performed for identification of different cells at the 3th day. To determine the number of cells expressing a particular antigen, 20 fields per sample were counted. Results are expressed as percentage for the total numbers of positive cells for MAP2 and GFAP to total cell numbers. Results are mean±SD of data of 10 samples from three independent experiments. Statistical analyses were carried out using Student's t-test between control group and other group with SPSS 13.0 statistical software. p <0.05 for the difference was statistical significant.Results: 1. Induction of neural differentiation of NSCs by N-CM: The growth state of cells in natural differentiation group was very poor and cell nucleus was pycnotic and obtrite. The neurons were scarce, small size. The processes were not obvious. The neurons moved closer distance and aggregation together. The astrocytes were multitude, large, flat morphology and more gathered in the center of neurospheres. These two cells differentiated from NSCs were majority. However, the growth state of cells in N-CM group was well and cell nucleus was uniform and few obtrite and decentralized distribution. The processes were long. The distribution of neurons is uniform. The quantity of neurons in N-CM group was more than that in natural differentiation group. Neurons migrate out from neurospheres was farther than that in natural differentiation group. The astrocytes were less than natural differentiation group. The astrocytes were slender processes and more decentralized distribution. The results showed that the proportion of neurons in N-CM (34.26±9.31%) group is higher than Neurobasal group (18.02±7.69%) (p<0.01). Whereas the proportion of astrocytes (36.92±10.14%) was lower than Neurobasal group (60.11±8.99%) (p<0.01). 2. Neural differentiation of NSCs in inhibitor groups: The growth state of cells in PD98059 group and SB203580 group was still well. The cell nucleus were homogeneous and a small amount of fragmentation have been seen. Compared with the control group, neurons were smaller and migrated the short distance, the processes were not obvious, migration from, yet uniform distribution. The quantity of neurons in those two groups was less than control group. The results showed that the proportion of neurons in PD98059 group (20.37±9.25%) and SB203580 group (22.14±8.47%), both were significantly lower than that of N-CM group (34.26±9.31%) (p<0.01).Compared with the control group, the astrocytes were large, flat, irregular shape, more processes and decentralized distribution. The quantity of astrocytes was more than that of control group. The results showed that the proportion of astrocytes in PD98059 group (47.43±11.45%) and SB203580 group (48.72±10.58%), both were significantly higher than N-CM group (36.92±10.14%) (p<0.05). However, compared with N-CM group, there was no significant difference of neurons and astrocytes shape, and quantity of distance migration statistics between Genistin group and the control group (p >0.05).Conclusion: The data presented show that: 1) The soluble molecules secreted by by BMSCs can induce NSCs to differentiate into high proportional neurons; 2) The soluble molecules secreted by BMSCs not only can regulate differentiation of NSCs, but also can affect cell morphology and behavior of neurons and astrocytes included cell morphous,processes length, migration activity and so on; 3) The soluble molecules secreted by BMSCs up-regulates the proportion of neurons and down-regulates the proportion of astrocytes probably via the ERK1/2 signal transduction pathway and p38 signal transduction pathway, but not SAPK/JNK signal transduction pathway. It may account for the mechanism of signal transduction in NSCs differentiation. |
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