| Bankground and SubjectiveIn traditional views, the generation of mammalian neuron only takes place in the time of embryo and a short time after birth, and the differentiation of neuron stops in other time. The dead neurons caused by all kinds of reasons are replaced by astrocytes, which caused the impossibility of the restoration of the function of central nervous system. The discovery of NSCs, which brings us into a new epoch of neural restoration, changes the concept of traditional neural restoration, which makes the restoration of damaged neural function and the cure of difficult diseases in central nervous system possible. Therefore, the cure of diseases in nervous system with NSCs has been the focus of much attention. Although there are multipotent NSCs in the brain, they could not generate enough differentiated progeny when brain is damaged because of the small amount of NSCs and the lack of enough positive stimulating signals in the brain; and it is of great difficulty to acquire NSCs from the adult brain as well as the clinical utility of NSCs from embryonic stem cells and fetal brain are restricted because of ethical, logic, and immune rejection problems, which have been severely limited the clinical use of NSCs. Therefore, it hasgreat sense to discover new sources of NSCs for basic and clinical studies in neuroscience field.Mesenchymal stem cells (MSCs) can tran-differentiate into multiple cell lineages and be isolated, cultured, and proliferated easily. These characteristics of MSCs make them ideal engineering cells in cell and gene therapies. Many experiments have been showing that bone mesenchymal stem cells (BMSCs) can be differentiated into neural stem cells. Not only bone marrow but also human umbilical cord blood and peripheral blood contain MSCs. Recent studies have shown that human umbilical cord blood mesenchymal stem cells (HUCBMSCs) can differentiated into terminal neural cells just as BMSCs.HUCBMSCs were separated, cultured, proliferated, marked in vitro and then implanted into the head of injured brains. Then the survival, migration, integration, and differentiation of the implanted HUCBMSCs were monitored in this experiment. Comparing with other groups, the effect of traumatic brain injury cured by transplantation of HUCBMSCs was evaluated by behavioral score and study and memory ability. Implanted HUCBMSCs were observed for long time and inspected differentiation by immunohistochemistry. This experiment may give experimental and academic theory base for the clinical use of HUCBMSCs as a new source of cells for cellular therapeutic for diseases in neural system.Methods: Mononuclear cells (MNCs) were prepared from heparinized human umbilicalcord blood (50~100ml) samples (donated from volunteers) by centrifugation over Lymphoprep (1.077 g/ml) and cultured in DMEM/F12 medium containing 20 % fetal bovine serum. The non-adherent cells were removed after 24 hours, and all the liquid was changed every 3 days. Phenotypic changes were monitored by light microscopy. HUCBMSCs were labeled for 30 min with fluorescein Hoechst 33258 before transplantation. The models of traumatic brain injury rats were established by applying a free-falling device hitting the rats' brain directly. A total of 90 adult healthy Wistar rats were randomly allocated into 4 groups, which are Sham, TBI, TBI + NS and TBI + HUCBMSCs. Group TBI + HUCBMSCs has 24 and other groups has 22 rats. Four groups were evaluated by behavior in 2 days and 7 days after transplantation, and detected study and memory ability by Y-maze in 2 week and 4 week after transplantation. Survival cellswere observed by fluorescent microscope in 2 days and 7 days after transplantation. Necrotic neurons of injured zone were counted in HE slice. GFAP, NSE were tested according to immunocytochemistry method in 2 weeks and 4 weeks after transplantation.Results: Cells were small and round at the beginning (24 hours), and then slowly turned into bigger and oval shape (4 days), and sprouted (7 days), clone which made of hundreds of HUCBMSCs can be seen at 3 weeks, and the sprouts stretched out and waved together just the same as neuron at 7 days of the second generation. When frozen slices of 2 days after transplantation were observed by fluorescent microscope, we found that the marked HUCBMSCs were giving out blue-white lights and the morphology was intact and cells were spreading along the two sides of needle canal. The nearer to canal? the denser to spread. When frozen slices of 7 days were observed, we found the distribution of cells near canal was sparser than that of 2 days. There was no significant difference (P >0.05) between TBI + NS group and TBI group and TBI + HUCBMSCs group in the score of rats behaviors on 2 days after transplantation. But there was significant difference (P <0.05) between TBI + HUCBMSCs group and TBI group and TBI + NS group in the score of rats behaviors on 7 days after transplantation. The number of necrotic neurons in TBI + HUCBMSCs group is less than that of control group in 2 weeks after transplantation. The evaluation of Y-maze showed that there was significant difference (P <0.05) between TBI + NS group and TBI + HUCBMSCs group when 2 weeks and 4 weeks after transplantation.Conclusions:1. HUCBMSCs can be well cultured in DMEM/F12 medium containing 15 to 20 percent fetal bovine serum in vitro.2. HUCBMSCs can survive and migrate to injured zone and differentiate in traumatic rat brain. Brain injured rats that have received HUCBMSCs transplantation were significant improved motor function scores and study-memory ability compared with control groups.3. HUCBMSCs in injured rat rain can protect neuron, then differentiate neuron and glial which repair injured nervous tissues and restore function.4. HUCBMSCs can be one of the cell sources for the treatment of neural systemdiseases.
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