Directional Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Oligodendrocytes Induced By The Combination Of Various Neurotrophic Factors

Objective: 1.Investigate the method of isolation and cultivation of rat bone marrowmesenchymal stem cells (mesenchymal stem cells, MSCs) in vitro, and observe the ability ofproliferation and differentiation in vitro as well as their biological characteristics, but alsostem cell-related markers on the cell surface were identified for laying the foundation forfurther study on bone marrow-derived mesenchymal stem cells.2.Investigate the feasibility of experimental conditions of bone marrow-derived mesenchymalstem cells induced by the insulin-like growth factor and cytokines differentiated tooligodendrocyte . Observe the positive rate of differentiated cells by laser scanning confocalmicroscope and select the optimal conditions for inducing cells.3.On the basis of two experiments, to observe the bone marrow-derived mesenchymal stemcells proliferation and differentiation process, to determine the role of IGF-1 inoligodendrocyte differentiation and give a certain degree of intervention measures to confirmfurther.Methods: 1 From the original cells, adherent screening method and trypsin digestion wereused for isolation and purification of mesenchymal stem cells .Inverted phase contrastmicroscope were used to observe cell morphology and growth characteristics in the process ofits proliferation, MTT method to map out the growth curve, immunohistochemical methodwere used to identify cell surface markers CD44 of stem cells. the fourth passage cells were respectively cultured in osteogenic,adipogenic and neuro—differentiation induction medium, the alkaline Phosphatase staining, oil red O staining and morphological observation, multi-differentiation potential of stem cells were confirmed.2.At passage 4, BMSCs were incubated in serum-free medium, supplemented with N2, 20 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor for 48 hours, and incubated in medium containing 500 ng/mL insulin-like growth factor 1 and N2 for 3 days. Morphological changes were observed using an phase contrast microscope. Semiquantitative RT-PCR was utilized to detect specific marker mRNA expression of oligodendrocytes. Western-blot Determination was utilized to detect basic protein in rat phospholipid (MBP).Using neuron marker anti-microtubule-associated protein, astrocyte marker anti-glial fibrillary acidic protein, oligodendrocyte marker anti-galactocerebroside, anti-myelin basic protein antibody, immunocytochemical staining was performed to detect the positive rate of the differentiation of BMSCs into oligodendrocytes. Select the best IGF-1 (Insulin growth factor-1) concentration in the culture medium divided doses.3.in accordance with BMP2 (bone morphogenetic proteins) inhibiting oligodendrocyte formation , in order to obtain the molecular mechanisms of IGF-1 inducing oligodendrocyte differentiation, we infer the effects of IGF-1 are the inhibition of BMP signaling pathway. To determine whether BMP2 inhibit MSCs differentiate to oligodendrocyte, The differentiation culture medium with different doses of BMP2 were used for 3 days and to collect cell-specific markers for rat phospholipid basic protein (MBP), galactose cerebroside (Glac) and take statistical treatment ,semi-quantitative RT-PCR detection, immunocytochemical staining,laser scanning confocal microscope .Then to detect the percentage of positive cells of bone marrow-derived mesenchymal stem cells to oligodendrocyte differentiation.Results: After isolation and culture , cells arranged spindle or polygonal.cells were S-shaped growth curve, population doubling time were 20 hours of osteoblasts. induced by the agent 14 days after induction, alkaline Phosphatase staining is positive results , suggesting that the cells differentiated into osteogenic direction, the isolation and culture of the cells were induced into a fat-induced cultivate 14, and the oil red O staining shows that a large number of red cells with lipid droplets, suggesting that the cells can divide to the direction of fat cells, the cultured cells into neurons induced by the agent 6 hours after induction, cells were typical neuron-like form of performance, suggesting that the cells can divide to the direction of nerve cells. CD44 antigen MSCs immunocytochemical staining showed positive results, the above results, the isolated and cultured the cells comply with the requirements of experiments with multi-differentiation potential of bone marrow-derived mesenchymal stem cells.2①bone marrow-derived mesenchymal stem cells to the oligodendrocyte-induced morphological differentiation in the process of change: After induction of differentiation, the majority of bone marrow-derived mesenchymal stem cells showed oligodendrocyte morphological characteristics of the cytoplasm to the nucleus to shrinkage, cell processes extend outward, the refractive index increased, with the number of cell processes extend to form a typical interconnected network structure.②oligodendrocyte-specific expression of mRNA markers: cells can be detected after differentiation phospholipid basic protein mRNA, cerebroside galactose specific mRNA bands.③oligodendrocyte positive rate: conditions in inducing differentiation, galactose cerebroside positive rate was 65%, phospholipid basic protein-positive rate of 45%, microtubule-associated protein 2-positive rate was 10%. Comparison with different concentrations of IGF-1 in differentiation medium,we found that culture medium contain 500μg / L IGF-1 concentration was the highest positive rate in oligodendrocyte markers.④Western-blot detection of alkaline phospholipid significantly increased the level of protein expression in the culture medium o contain 500μg / L IGF-1 concentration.3 the differentiation culture medium by adding different concentrations of BMP2, was found that oligodendrocyte differentiation were inhibited, only part of bone marrow-derived mesenchymal stem cells showed oligodendrocyte morphological characteristics, and as dependent on the concentration of BMP2. the relationship between the higher concentration (5μg/L-50μg/L), the less of oligodendrocyte marker (Glac, MBP)-positive cells .charged cell-specific markers for rat phospholipid basic protein (MBP), semi - galactose cerebroside (Glac) semi-quantitative RT-PCR detection,we did not detect specific bands. At low concentrations (0.05μg/L-0.5μg/L) circumstances, it can be observed in some cells to oligodendrocyte differentiation, the results consistent with BMPs inhibitting oligodendrocyte differentiation characteristics.Conclusions: take tissue culture of rat bone marrow, adherent with screening, trypsin digestion and purification, MSCs can be isolated with a stable number of CD44 antigens to thedifferentiation potential of bone marrow-derived mesenchymal stem cells, the cells continuedto mesodermal cell differentiation into, but also to the ectodermal neural cell differentiation, itwas an ideal cell to study directed differentiation of stem cells in vitro.2.The combination of epidermal growth factor, basic fibroblast growth factor and insulin-likegrowth factor can effectively promote the directional differentiation of BMSCs intooligodendrocytes. We grasp the experiment conditions of bone marrow-derived mesenchymalstem cells to oligodendrocyte differentiation and lay the foundation for the treatment ofspinal cord injury by subsequent mesenchymal stem cells to the oligodendrocytedifferentiation transplantation.3.the main mechanism of insulin-like growth factor-induced bone marrow-derivedmesenchymal stem cells to oligodendrocyte differentiation, are to inhibit the signal path ofspecific BMPs inhibitory factor of oligodendrocyte precursors in the spinal cord .