| ObjectiveIn this theses, we were going to explore the effect ofβ-Mercaptoethanol (β-ME) on rat bone marrow mesenchymal stem cells (MSCs) towards the direction of neural cells,and to investigate the function of Notch singnaling during the process of MSCs'neural cell differentiation, in order to clarify the mechanism of neural differentiation induced byβ-ME.Methods1. Isolation and culture of MSCs : the bilateral femurs and tibias of adult male SD rats were obtained under sterile conditions. Bone marrow cavity was washed with 5ml L-DMEM. The rinse solution containing the bone marrow treated with percoll separation and centrifugal separation,cells were counted and adjusted to the density of 1×109 /L,the MSCs were cultured in the culture box filled with 50 mL / L (volume percentage) of carbon dioxide and constant 37℃temperature .2. Neural differentiation of MSCs: After the cells spread to the five generations L-DMEM+1mmol/Lβ-mercaptoethanol +20% FBS were added in the culture flasks, and after 24hours L-DMEM +5 mmol/Lβ-mercaptoethanol were used to start neural differentiation. According to different induction time, MSCs were divided into 3 groups : A group:control group, pure culture; B group: 1 hour induction group; C group: induction 5 hours group. To take photos in the inverted phase contrast microscope before and after induction. 3. Identification of nerve cells: The cells before and after induction were detected by immunohistochemical SP method to observe the expression of neural precursor cell-specific markers including neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and nestin (Nestin), The cells with brown grains were judged positive, without any colours were negative.4. Transmission electron microscope were used to observe the ultrastructure of induced MSCs.5. Nerve-specific phenotype were detected by western blot .6. Notch signaling pathway detection: The total mRNA of the four groups cells were isolated before and after induction,to mensurate the purity with ultraviolet radiation spectrophotometer. The Notch1,Jagged1,Hes1 and Hes5 mRNA of three groups were expanded by realtime reverse transcription polymerase chain reaction (RT-PCR). To analysis the results with SPSS ver 13.0.Results1. Most cells of four groups adhered to the flasks and appeared long shuttle-like. Their character was stable and the morphologies of MSCs had no change after passage but increased more quickly.2. After induced byβ-ME+FBS, the cells body of most MSCs contract and exhibit spherical or pyramidal, with increasing thin processes, some induced cells had the neuron-like morphology. More and more cells changed after 5 hours, the cells emitted two or many poles and interlaced together, just like the typical neurons.3. The results of the immunocytochemistry indicated: the NSE,NESTIN and GFAP showed positive stains of three groups cells, which meaned the three groups cells differentiated into neuron. 4. The MSCs after induction were observed by transmission electron microscope, we founded that neuron-like cells'nucleuses were big ,global with obvious nucleolus. They were rich in organelles. The cytoplasm showed obvious golgimplex, rough endoplasmic reticula and numerous mitochondria.5. The neural-specific phenotypic expression of induced MSCs were detected by westernblot: compared with A group control, B group and C group showed a increased expression of NSE and NESTIN protein .6. Real-time PCR results showed that: three groups of cells expressed Notch1, Jagged1, Hes1, Hes5. There was a statistical significance in the MSCs before and after induction .Conclusion1. The whole bone marrow-adherent and density gradient centrifugation can get a higher dynamic MSCs, passage and exchange of fluid method can be purified MSCs successfully and established a stable rat bone marrow-derived mesenchymal stem cells proliferation system.2. The morphology, ultrastructure and neuronal cell-specific phenotype of protein identification, in this experiment under the conditions of culture induction, MSCs can be induced to differentiate into neurons in vitro.3. MSCs induced to differentiate into neuronal cells in the process, accompanied by Notch signaling pathway-related genetic alteration, suggesting that Notch signaling pathway involved in the direction of MSCs into neuron-driven differentiation.
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