| ObjectiveSeveral researches explored the stem cells. Recently, Studies of stem cells have suggested the existence of another kind of stem cells, besides of embryonic ones, which have the ability of self - renewing and differentiation. Generally, they were called adult stem cells. Mesenchymal stem cell( MSCs) is the kind of ones, which can be found in bone marrow. With differentiate induced conditions , MSCs can differentiate into a variety of non - mesenchymal cells, including osteoblasts,chondroblast ,adipocytes, nerve cells ,and so on. Generally, it is easier for both separation , cultivation, proliferation and introduction and expression of exogenous gene. In addition, MSCs have be expected to be new target cells of cell and gene therapy, our studies focus on the possibility of improving the recovery of injured spinal cord by means of implantation of cultivated MSCs in vitro. On basis of mention , we investigate the ability of isolation, purification and expansion of adult rat mesenchymal stem cells( MSCs) to nerve cells in vitro.MethodsBone marrow mononuclear cells from adult rat femur and tibia were obtained sterilely by Percoll ( 1. 073 103 g/L ) gradient centrifugation . The low density cells including MSCs were cultivated and expanded in MSCGM media . they were passaged two to ten times by being detached and replatedat a density of 0.4xlOVml. Expanded MSCs were induced to differentiate into nerve cells withthree different treatment protocol in the prensent study . On the following day, the medium was replaced with preinduction medium consisting of DMEM, 20% FCS, and 10 ng/ml basic fibroblast growth factor. After 24 h, the preinduction medium was removed, the cells were washed twice with PBS, and neuronal induction medium containing DMEM supplemented with 2% DMSO and200 m bu-tylated hydroxyanisole was added. In later experiments, we used DMEM with 5 mM p - mercaptoethanol or sulfo - glycerine as an alternative neuronal induction medium for the same incubation. Three control groups were set up. Neuron -specific enolase ( NSE ) , neurofilament ( NF ) , glial fibrillary acidic protein (GFAP) and Nestin were detectedby immunocytochemistry , and analysis of quantitation of neuronal differentiation. For quantitation, a Fugix digital camera was used to capture 10 non - overlapping low power images of each sample. The mean and standard deviation were computed.ResultsAdult rat MSCs were successfully cultivated . Under inverted microscope observing, About 2d after plating, primary cells adherent to culture dish, supernatant containing nonadherentcells was removed and fresh medium was added, to appear cell colony in the 5d ~ 6d or so, After the 10d ~ 12d the cells had grown to near confluency. Passage cells distribute uniformly , after 10 passages cell reproductive activity get weaken, To facilitate long - term survival of MSC, bFGF one of several additional components were added to the culture media. All three treatment protocol can induce MSCs differentiate into neuron - like cells in the present study . With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF - M. The retractile cell bodies extended long processes terminating in typical growth cones and filopodia. The neuron - like cells express NSE, NF and Nestin, but could not express GFAP. The majority of MSCs treated in those manner exhibited neuronal morphologies and stained positive for NSE(76.3 2. 1) % ,NF(78.22.5) % and Nestin (49.79 2. 3) % expression . Among the three methods, we found that No. 1 one excelled the two others, to be observed for seven days, it still existed 10% living cells.ConclusionsThe MSCs described here have the ability to proliferate extensively, and they maintain the abihty to differentiate into multiple cell types in vitro, and to offer reference for the application of MSCs in the field of neuroscience .
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