| Back GroundThe midbrain is a main part of brain stem. Its superior part is diencephalon, and lower part is pons. The mesencephalon is complicatedly composed of much nucleus and fibrous bands. Midbrain injury is a special type of brain stem injuries, which is often accompanied injuries of other brain parts. About 50%~70% of brain stem injuries accompanied various midbrain injuries. Primary brain stem injury is a very seriously fatal injuries. Its mortality is as high as 44.1%~62.5%, and its disability rate of survivor reach to 60%, and vegetable state patients surpass 1/3 of them. How to make the diseases of central nervous system, include PVS, paraplegia etc., obtain appropriate therapy, to recover well, and to make its outcomes, sequelaes and complications decreasing to minimum level, has been constantly the target of all neurologists, surgeons and neuroscientists.The investigations of recent years manifest that nervous system have the auto prosthetic potential. It has been discovered that in adult nervous system remained little neural stem cells, which could proliferate in some situations. Therefore, the latest and hopeful methods, for the treatment of trauma and regression diseases of nervous system, are through artificial intervention and controlling of the endogenous neural stem cells re-proliferation and differentiation, or proceeded to the transplantation with neural stem cells to promote the plerosis of the central nervous system injuries.The discovery of the multi-potential of bone marrow stromal cells and the successful oriented differentiation towards neural cells, which increase a new source of neural stem cells, and also provide a new idea for resolving the problems of the quantity and resource of neural stem cells in the auto-transplantation project.By means of in vitro and in vivo model, to investigate the projects of the purification, amplification and differentiation and the situations of the survival, migration, integration of bone marrow stromal cells, which is usefulfor the standardized develop and apply of the "neural stem cells derived from bone marrow stromal cells" in clinic.Part OneObjective1. To establish the method of high-speed bone marrow stromal cells amplification in vitro, for the extensive applying of bone marrow stromal cells serving as "seed cells" in the fields of medical science and tissues engineering.2. To set up model of bone marrow stromal cells orientation differentiation into neural stem cells in vitro, and to quest the feasibility of differentiating into neural stem cells from bone marrow stromal cells, and to provide theory base for the clinical application of neural stem cells derived from bone marrow stromal cells.Methods1. Isolation and amplification culture of bone marrow stromal cells Acquired bone marrow under aseptic condition; Density gradientcentrifugation; Adherence Technique to acquire bone marrow stromal cells; Then added bFGF(10ng/ml), EGF(10ng/ml) into D-MEM/F12 culture medium(l ml/hole); Two weeks later, trypsin method digested, and collected cells. The harvested cells was produced into 104 alive cells/ml density for later using; Then the cells was diluted 50 times with D-MEM/F12 and continued to amplification.2. Influence of bFGF and EOF on adhesiveness of BMSCsAdded different end concentration bFGF and EGF alone or associatively, into culture plate after work cells had been seeded into 96 hole tissue-culture plate and hatched for 6 hours; Then BMSCs cells were fixed with 2% glutaraldehyde and counted. There is difference between the effects of bFGF and EGF on BMSCs adhesiveness.3. Effect of bFGF and EGF on survival and proliferation of BMSCs Determined the effects of bFGF and EGF on the level of survival andproliferation of BMSCs by routine MTT and clone count methods at 1@24,3 @24h, 5@24h, 7@24h.4. The influence of RA, BDNF and GDNF on oriented differentiation of BMSCsSABC Technique was used to identify the cells state of differentiation under various concentrations of RA, BDNF,...
|
PDF Full Text Request