| ObjectiveNeonatal hypoxic-ischemic encephalopathy (hypoxic-ischemic brain damage, HIBD) is a degeneration of nerve cells induced by a variety of reasons,lead to degeneration and loss of nerve cells, vesicles of the organization, structure and function of the brain caused by the damage lead to permanent neurological damage, movement, learning and memory impairment, resulting in a number of serious complications.It is an important reason that lead to cerebral palsy (incidence of 3%o-5%o). In recent years, with the improvement of neonatal rescue techniques, the incidence rate was gradually increased,but the current therapies efficacy was poor. An urgent need to find a new rational and effective treatment measures, alternate damaged nerve cells.Derived from human umbilical cord blood mononuclear cells (human umbilical cord blood mononuclear cells UBC-MNCs) of neural stem cells (neural stem cells NSCs) are immature neural precursor cells, with ability of self-renewal and multilineage differentiation potential, under certain conditions, may be proliferate and differentiate into neurons, astrocytes and oligodendrocytes. NSCs transplantation can replace damaged nerve cells, integrate into the neural circuits, improve the animal's nervous function, it is an effective means of cell renewal, new treatment ideas and approaches of HIBD.Impact of NSCs proliferation and directional differentiation of factors that are very complex, can be attributed to both internal and external causes. Internal factors are mainly self-regulation of genes, such as Foxgl gene and Nestin genes, they induce cell proliferation and differentiation by activating the relevant transcription factors, activating or inhibiting gene transcription. External factors are mainly cytokines and the microenvironment in the regulation of the role of external signals, such as hEGF, bFGF, and B27 factors, in the micro-environment, the various components of extracellular matrix by regulating the adhesion and migration capabilities, as well as combined in a variety of extracellular matrix, growth factors and cytokines in the interaction, affecting the proliferation and differentiation of NSCs.In this study, cultured cord blood-derived NSCs in vitro, research the key proliferation and differentiation regulators of Nestin and Foxgl of NSCs, Nestin and Foxgl expression of their mRNA and the relationship between them. BrdU marked the NSCs then intravenous transplanted into HIBD newborn rats. From the histological performance, behavioral performance and in terms of learning and memory abilities of the HIBD newborn rats to evaluate the efficacy. The feasibility and timeliness of NSCs transplantation in the treatment of neonatal rats HIBD were discussed. It provide a more adequate theory of NSCs clinical transplantation in the treatment of neonatal HIBD.Methods1. The UBC-MNCs were isolated from human umbilical cord blood with density gradient centrifugation method, cultured in Neurobasal medium adding with hEGF,bFGF and B27 factures in vitro, the UBC-MNCs were inducted and diferentiated into NSCs. The characteristics of NSCs morphology and proliferation and diferentiation were observed. Culturing cells specific neuron antigen markers of Nestin,NSE,GFAP were detected by immunohistochemistry.The cultured cell's mRNA expressions of Foxgl gene and Nestin gene were performed by reverse transcription polymerase chain reaction method.2. Target cells were traced by BrdU and cell proliferation indexs were determined.The cultured NSCs were injected intravenously into neonatal rats encountered cerebral hypoxic-ischemic damage.The expresstions of Nestin,NSE,GFAP,BrdU positive cells in SVZa areas which are damage vulnerable brain areas and NSCs survival,migration and differentiation were observed by immunohistochemistry at 1d,7d,14d,21d after transplantation,pathological analysis were done at the same time by hematoxylin-eosin stain.The neurological assessments and Y maze tests were respectively carried out at 1d,7d,14d,21d after transplantation,the therapeutic effect of cell transplantation on HIBD neonatal rats was observed.Result1. The UBC-MNCs can differentiated into neuron-like cells after orientid induction,cultured cells can grow into typical NSCs cloning balls,express labelled antigens of Nestin,NSE and GFAP, before induction, the cells expressions of Nestin, NSE, GFAP were very low, Nestin positive cells came to its peak at sixth day after induced, then begin to decline, NSE and GFAP positive cells gradually increased after induced. Positive BrdU labeled target cells were 90%.2. The cell's expression of Foxgl gene mRNA and Nestin gene mRNA low before inducted,then increased gradually after inducted(P<0.01), came to its peak at 6 days, then declined gradually (P<0.05). Nestin gene mRNA increased gradually (P<0.05).The cell's expression of Nestin gene mRNA were always lower compere to the expression of Foxgl mRNA at the time of 3d,6d,9d,12d after inducted.3. The expressions of Nestin-positive cells in SVZa area of all the animals increased early and then decreased, NSCs transplantation group increased rapidly after transplantation, came to peak at 14d, then decreased gradually.The expressions of NSE-positive cells in SVZa area of normal control group and NSCs transplantation group general increased gradually, NSCs transplantation group, which increased rapidly, the model control group decreased gradually.The comparison of the expressions of Nestin-positive cells and NSE-positive cells in SVZa area after transplantation 1d hours among the three groups, P values were P>0.05, there were no significant statistice differences.The expressions of GFAP-positive cells in SVZa areas of all the animals increased gradually,the highest is the model group,and the lowest is the NSCs transplantation group.BrdU-positive cells were observed in SVZa area in NSCs transplantation group,they could chemoattractanted to the left SVZa areas and survived, migrated from the surrounding area to the central ischemic necrosision,the damage area decreased significantly compared with the model group.With the prolonging of cell transplantation, the expression of BrdU-positive cells was growing, P<0.05, there were significant statistic differences.4. The animals learning and memory evaluation by Y maze test showed:at the aspect of learning,with the age increased, the learning ability of NSCs transplantation group, model control group, normal control group increased gradually. The learning and memory ability decreased significantly after HIBD (P<0.05). There were no significant learning and memory differences between there groups at 1d after transplantation, but the differences between transplant group and normal control group at 7d,14d,21d after transplantation (P<0.05).Conclusion1. We can obtain NSCs from UBC-MNCs that were inducted and cultured in vitro,human cord blood can be the new source of neural stem cells.2. Foxgl gene and Nestin gene are the key proliferation and diferentiation regulation factors of NSCs.3. NSCs transplantion could improve the rehabilitation of organizational structure and reconstruction of cell functions in SVZa areas, promote the learning and memory ability of HIBD neonatal rats, impetus their behavioral function recovery.4. The NSCs transplantation can inhibit the physical disorders continue to develop after HIBD injury in neonatal rats, promote growth and development in rats.5. NSCs transplantation may be a new approach to treatment of HIBD.
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