Protein Microarray Analysis Of Protein That BMSCs Regulate The Differentiation Of NSCs

Objective : Neural stem cells (NSCs) can differentiate into neurons, astrocytes and oligodendrocytes and have the ability of self-renewal, and can provide tissue cells for brain. For the past few years, scientists found that the adult central and peripheral nervous system are both have NSCs, this findings brings advan- tage for neural degenerative disease such as Parkinson's disease, Alzheimer disease and nervous system damage, for example cerebral trauma. While neither in vitro nor in vivo, the percentage of neuron are much lower than glial cell in the offspring of NSCs, thus the neurons are not enough to substitute those lost in brain damage and disease. Bone Marrow Stromal cells (BMSCs) are multipotential stem cells existing in bone marrow, which have the characteristics of adherement and clonal reduplication in vitro culture, and the main function of them is provide structural and functional support for hematopoiesis. In recent years, BMSCs were found that can excrete many neural protective and prosthetic cytokines, such as brain-derived neurotrophic factor, basic fibroblast growth factor, vascular endothelial growth factor, interleukins, transforming growth factor et al, further more can affect the survival, multiplication and differentiation of NSCs. Some researches confirmed that BMSCs can directly regulate the differentiation of NSCs into high proportional neurons, furthermore the dissolvable molecules excreted by BMSCs play a key role during this process, which were surpported by the study of Bone Marrow Stromal cells conditioned medium (BMSCs-CM).While what kind of dissolvable molecules play a part in this process is still unknown. Using the BMSCs conditioned medium(Neurobasal Conditioned Medium, N-CM) after segmentation to culture NSCs, preliminary screening the part that can regulate the differentiation of NSCs, then the useful part was detected by Ray Biotech Rat Cytokine Antibody Array. The research intend to reveal the mechanism that BMSCs regulate the differentiation of NSCs.Methods: The routine methods were used to extract BMSCs from Sprague-Dawley adolescent rats and mesencephal NSCs from neonate rats . Then the BMSCs from 3 to 6 passages were planted in culture flask of 25ml, changed the culture medium for 6 ml Neurobasal medium when the cells spread 85% area of the bottom. The Neurobasal Medium was collected after 24h and taken as Neurobasal conditioned medium(N-CM), then was storaged in -800C refrigerator after centrifuge. The N-CM was divided into two parts by concentrated through a 5KDa ultrafiltration membrane (the cut-off molecular weight is 5KDa). One part contained the protein which molecular weight was above 5KDa, another was bellow 5KDa. There were 4 groups: (1) Neurobasal group. (2) N-CM group. (3) N-CM>5KDa group.(4) N-CM<5KDa group. Cellular immunofluorescence staining was executed in the third day after differentiation. The differentiated cells were observed by fluorescence microscope and counted. The selected useful part was detected by Ray Biotech Rat Cytokine Antibody Array. Statistics analyses were performed by SPSS 13.0 software. Results were expressed as mean±standard deviation. Independent T Test was used in this study.Result: 1.The impact of different culture solution on the differentiation of NSCs: (1) Neurobasal group: The cells were not in good condition, many cellular nucleus shrinked and broke into pieces. MAP-2 positive neurons were fewer and most assembled around neurospheres. The ecptoma of neurons and NG2 positive oligodendrocytes were short. There were more GFAP positive astrocytes than other groups. (2) N-CM group: The total state was better than Neurobasal group. Most of the cellular nucleus were intact. The MAP-2 positive neurons were more and the stain were deeper compared with Neurobasal group. The ecptoma of neurons and oligodendrocytes were longer than those in Neurobasal group. (3) N-CM>5KDa group: The cells growth state was the best. Most cellular nucleus were equal in size and intact. The MAP-2 positive neurons took up most of the offsprings. The ecptoma of neurons and NG2 positive oligodendrocytes were long. There were less GFAP positive astrocytes than other groups.(4) N-CM<5KDa group: The state of cells was extremely bad, most of the offsprings dead in 3 days. 2. The results of cell counting and statistic analyses: (1) The quantity of MAP-2 positive neurons: The percentage of MAP-2 positive neurons in N-CM group (22.16±9.11%) was higher than that in Neurobasal group (17.00±7.69% p=0.021, p<0.05), which indicated that N-CM promoted the NSCs to differentiate into neurons. The percentage of MAP-2 positive neurons in N-CM>5KDa group (34.24±15.94%) was higher than N-CM group (p=0.0006, p<0.01), which implied that the fraction of N-CM>5KDa play a dominate effect in this process. There was no data of N-CM<5KDa group because most of cells had dead when staining.(2)The quantity of NG2 positive oligodendrocytes: The percentage of NG2 positive oligodendrocytes in N-CM group (25.82±7.79%) was higher than that in Neurobasal group (21.32±9.09%) (p=0.044, p<0.05); the N-CM> 5KDa group (41.01±8.88%) was higher than N-CM group (p=0.000, p<0.01), which illustrated that N-CM could regulate NSCs to differentiate into more oligodendrocytes and the molecular weight above 5KDa played the main role. (3)The quantity of GFAP positive astrocytes: The percentage of GFAP positive astrocytes in N-CM>5KDa group (23.10±13.57%) was lower than that in Neurobasal group (37.99±10.75% p=0.000, p<0.01) and N-CM group (35.07±8.96%, p=0.001, p<0.01), but the difference between the other two groups was no significant, which indicate that N-CM>5KDa could inhibit NSCs to differentiate into astrocytes. 3. The result of Rat Cytokine Antibody Array: The medium of N-CM>5KDa was detected by the Rat Cytokine Antibody Array, seven cytokines were detected up-regulated 1.5 times in N-CM-above-5KDa compared with the Nerobasal Medium (molecular weight above 5KDa), such as cytokine-induced neutrophil chemoattractant-3 (CINC-3), cholinergic neuronotrophic factor (CNTF), immunoreactive fibronectin (IFN-γ), interleukin-1α(IL-1α), monocyte chemoattractant protein (MCP-1) , tissue inhibitor of metalloproteinase (TIMP-1) and vessel endothelium growth factor (VEGF), There was no cytokine down-regulated .Conclusion: The research aimed at approach the mechanism that BMSCs regulate the differentiation of NSCs by using Neurobasal medium as conditioned medium of BMSCs. The result manifested: (1) The dissolvable molecules secreted by BMSCs can promote NSCs to differentiate into more neurons and oligodendrocytes and promote their mature, as the same time can inhibit NSCs to differentiate into astrocytes, in which the molecular weight above 5KDa play a key role in this process. (2) The 7 cytokines such as CINC-3, CNTF, IFN-γdetected by protein microarray analysis might play a key role in this regulate contribution.(3) The dissolvable molecules secreted by BMSCs can promote the offspring neurons of NSCs to migrate.