Influence Of Movement Training On Transplantation Of Neural Stem Cells In Rats With Middle Cerebral Artery Occlusion

PartⅠThe culture and identification of neural stem cellsObjective:To master the technology of neural stem cell culture and provide neural stem cells for transplantation in the following experiments.Methods:We chose SD rats embryonic cerebral cortex and hippocampus of embryo day12～14 as the resource to culture the neural stem cells.The tissue was isolated and mechanically broken into single cell suspensions.The suspensions was centrifuged and the sediment was re-suspensed with neural stem cell culture medium,in accordance with the density of 1×10~6cells/ml.Then the cells was inoculated in culture bottles and observed regularly.The medium was semi-changed with interval of 2-3 days and the cell was passaged at the interval of 7-10 days.The cultured cells were then identified for proliferative activity,differentiation capacity and neural stem cell line-specific antigen nestin.Results:Cultured neural stem cells gathered into a ball of cells and grown in a suspended way.7-10 day after the inoculation,the dense cell ball of uniform size was formed and they are still available even after many times passages.The detection of nestin and BrdU of cultured cells were positive and they can aslo differentiate intoβ-Tublin or GFAP-positive cells.Conclusion:The cultured cells of this part of experiments have the ability of self-replicating and multi-directional differentiation,which is consistent with the requirements of neural stem cells for transplantation.PartⅡThe study of antigen expression in neural stem cells in vitro differentiationObjective:To explore the trends of Nestin,SOX2,DCX,TuJ1,GFAP,NeuN expressed in the process of differentiation from in vitro neural stem cells into neurons and to lay the foundation for further studies concerned with neural stem cell proliferation and differentiation factors as well as in vivo transplantation.Methods:We chose embryo day12～14 of embryonic cerebral cortex as the resource to culture the neural stem cells in vitro;Three days after the 1st passage,the neural stem cells were induced to adherence to cover glass and differntiation in specific medium;Immunofluorescence methods were used to detect nestin,SOX2,DCX,TuJ1,GFAP,NeuN at different timepoints(day1,day4,day7,day10,day14).Results:The expression of SOX2 and Nestin at the most of the cells at the beginning are similar,the expression of both Nestin and SOX2 gradually decreased with the passage of time,but the expression of sox2 lasted a relatively longer term;High expression of DCX in day1,day4 and day7,and then began to decrease,at day14,the differentiated cells were only a few DCX-positive;Only a small number of TuJ1 positive cells in the day7,then followed a obviously increased in its expression. Day14 has 38.27 percent of the cells to express TuJ1.No NeuN positive cells advented in day1,day4 and day7,while at day14 has been NeuN positive cells accounted for 21.11 percent.GFAP-positive cells crossed a whole process of differentiation,high expresstion at the first week,day10 and day14 were still a about a half of total cells being GFAP-positive cells.Conclusion:Expression of specific antigens in the process of differentiation in neural stem cells is a complex process,during which the expression of antigens that represent undifferentiated state of cells reduces,while that of antigens representing differentiated state increases,both are consistent with each other.PartⅢThe role of treadmill training in rats of ischemic stroke in the treatment of neural stem cells transplantationObjective:To study the influence of treadmill training after neural stem cells transplantation on the neural function and cerebral ultrastructure of rats with focal cerebral ischemia.Method:Select middle cerebral artery ischemia / reperfusion Sprague-Dawley rats as subjects.The subjects are divided into control and treatment(including:treadmill training,neural stem cells transplantation,neural stem cells transplantation combined with treadmill training) groups,each with 6 or 10 animals.Five days after opration, neural stem cells marked with SPIO(superparamagnetic iron oxide) were transplanted into ischemic striatum and electric treadmill training begun 6 days after operation.For all groups during four weeks of training,assessment of motor function are conducted regularly.Four weeks after training,rats were sacrificed and SPIO marked neural stem cells were observed in terms of survival and movement.Host ultrastructural changes are aslo observed.Results:Compared with the control group,there is a of significant difference in both treadmill training group and combined group(P＜0.05,P＜0.01) concemd with mNSS 14 days after the training while the mNSS of neural stem cells transplantation group is not of significant difference(P＞0.05).The SPIO marked neural stem cells in the combined group had a greater density and spread more extensively,and their cerebral ultrastructure are better than other groups.Conclusion:Treadmill training combined with neural stem cell transplantation is of potential benefit in the treatment of rats with cerebral infarction,but the mechanism underlaying this effect need to be studied further.