| ObjectiveThe discovery of neural stem cells and the establishment of the isolation, i-dentification and culture technologies for them not only give a great challenge to the traditional theory that neural cells couldnt be regenerated, but also open up a new way for the treatment of the diseases of the nervous system. But the use of neural stem cells is severely limited by some factors, including the little amount and dispersed distribution of neural stem cells, the difficulties in obtaining materials for culture in vitro and setting up the immunogenicity - free neural stem cell system, the difference of categories in cross transplantation, and sociological and ethical problems. With the intensification of the researches concerned, stem cells used for transplantation can now be obtained from many sources. There are some medulla stromal stem cells ( MSCs) in the medulla of adult animals. Recent information shows that the differentiation of medulla stromal stem cells towards neural stem cells, neurons and neuroglia can be realized under appropriate conditions. Some breakthroughs have been achieved in the induced differentiation of MSCs to neuron - like cells, but these induced neuron - like cells cannot survive long, most of them will die in 24 hours. So it's extremely important how to prolong the life of MSCs - born neuron - like cells. Some achievements have been made in the treatment of ischemic brain damage by MSCs transplantation. However, after transplantation, more MSCs are differentiated to neuroglia but not neuron - like cells in vivo. Therefore, much better results will be obtained if medulla stromal stem cells are first differentiated to neuron — like cellsin vitro and then transplanted.In the experiments, rat medulla stromal stem cells are isolated, cultured and purified by the in vitro cell culture technology, their activities, growth curves and attachment efficiencies are determined, and the cultured medulla stromal stem cells are identified morphologically. These cells are subcultured and expanded in vitro to give some cells, which are intervened with melatonin ( MT) and bFGF to prolong the survival time of the induced neuron - like cells, and MSCs - born neuron - like cells that survive longer are used for transplantation in the model of cerebral artery occlusion (MCAO) of rats. The distribution and survival of the implanted cells are observed, and the influence of the implanted cells on the expression of the MCAO cerebral neuron C - myc, Erk Protein and mRNA is studied.MethodsObjectiveA. The isolation, culture, expansion and purification of rat medulla stromal stem cells1. The isolation of rat medulla stromal stem cells: rat medulla stromal stem cells were isolated by attachment and density centrifugation.2. The expansion and purification of rat medulla stromal stem cells: an expansion and purification system was set up for rat medulla stromal stem cells to provide seed cells for the experiments.3. The activities of the cells were determined by Trypan Blue.4. The attachment efficiencies of the cells were determined.5. The identification of medulla stromal stem cells: the surface markers CD45 and CD90 of the cultured cells were identified by immunofluorescence.B. The researches on the induced differentiation of rat medulla stromal stem cells towards neural cells1. The MSCs were cultured and purified.2. The oriented induced differentiation of MSCs towards neural cells: with MT and bFGF, rat medulla stromal stem cells that had been cultured in vitro were induced to differentiate to neural cells.3. The identification of induced cells by immunofluorescence: the expression of the differentiated cells NSC. E and GFAP was identified by immunofluorescence.1. The researches on the treatment of focal cerebral ischemia of rats by the transplantation of differentiated rat medulla stromal stem cells2. Rat medulla stromal stem cells were isolated, cultured and expanded.3. The induced differentiation in vitro of MSCs to neuron — like cells was conducted. After differentiation, the cells were marked with BrdU in vitro.4. The model of cerebral artery occlusion ( MCAO) of rats was made and the functions of nerves were graded.5. MSCs - born neuron - like cells were injected into the MCAO via the carotid.6. The immunohistochemical treatment of the BrdU - marked cells: the survival rate of the implanted cells was observed.7. C - myc and Erk in the brain of rats were determined by the immunohistochemical technique, and C - myc, Erk and mRNA were determined by in situ hybridization. The effect of implantation of cells on these indexes was investigated.ResultsA. The isolation, culture, expansion and purification of rat medulla stromal stem cells1. One week after the inoculation, cell colonies increased rapidly. The colonies continuously expanded and mixed with each other, and fully attached to the bottom of the culture bottle in 10 -12 days. The cells mainly took the shapes of shuttle and circle.2. The subcultured cells didnt grow in colonies any more. They grew faster than the primary cells.3. No remarkable difference was observed in the attachment efficiencies of PI, P3 and P5 cells of MSCs at various times.4. The inoculation period of the subcultured MSCs was 1-2 days. From the3rd day on, the cells greatly proliferated in log phase. The proliferation reached the fastigium on the 6th day and entered the plateau after that.5. MSCs expressed CD90 and CD44 but not the surface markers CD34 and CD45 of hematopoietic cells.B. The researches on the induced differentiation of rat medulla stromal stem cells towards neural cells1. Three days after the cultivation liquid containing MT and bFGF was added , a part of the neurons were found to contract, refract more light and protrude and bulge. Some neurons contracted to a triangular shape, and some others became simple bi - polar or multi - polar cells.2. After being induced for 72 hours, the cells began to express NSE. Positive cells increased in 9 - 14 days with the MT + bFGF group changing remarkably, but didnt express GFAP.3. Erk Protein was expressed weakly in MSCs cytoplasm before the induced differentiation. However, 3 days after being induced, a great amount of Erk positive cells appeared.C. The experiments and researches on the treatment of focal cerebral ischemia of rats by the transplantation of differentiated rat medulla stromal stem cells1. Grading conducted showed that the functions of nerves of the MCAO rats with the cells implanted were evidently improved compared with the control.2. Forty - eight hours after the implantation of the cells, dispersed BrdU positive cells were found in the damaged brain of rats. Seven days later, BrdU positive cells gradually increased but didnt increase any more from the 14th day on.3. Forty - eight hours after the implantation of the cells, C - myc mRNA positive cells could be found in the basal ganglia, cortex and hippocampal neurons of the MCAO rats.4. Twenty - four to forty - eight hours after the implantation of the cells, Erk Protein and Erk m - RNA positive cells increased in the brain of the MCAO rats. One week later, Erk Protein positive cells still remained at a high level.Conclusion1. Density gradient centrifiigation and attachment culture are simple and efficient methods in the isolation of medulla stromal stem cells. The great expansion of MSCs is possible. MSCs express CD90 and CD44 surfacely, and the rate of positive cells is high.2. A majority of the MT and bFGF induced neuron - like cells can survive for 14 days and stably express the specific marker NSE of neuron - like cells. The activation of Erk signaling pathway by MT and bFGF may be one of the mechanisms of the oriented induced differentiation of MSCs.3. The implantation of neuron - like cells via the carotid remarkably improves the functions of nerves of the MCAO rats. The implanted cells may inhibit the apoptosis of neurons by decreasing the C — myc level and activate Erk signaling pathway to promote the recovery and regeneration of damaged neural cells.
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