Bone Marrow Stromal Cells Differentiate Into Neurons And Treatment Of PD Rat With The Differentiated Cells

Objective: Bone marrow stromal cells (BMSCs) can differentiate into bone, cartilage, muscle, neuron, and glia both in vivo and in vitro. Recently, BMSCs differentiated into neuron and treated nervous system diseases have become the focus. But the neurons death very soon used the methods from the initial report. So, It is difficult to do more research in the differentiated cells' function. In the experiments here, we used ascorbic acid, adult cerebrospinal fluid (CSF) to induce BMSCs differentiating into neurons. And used several factors (BDNF, NT-3, PDGF, IL-1, IL-11,LIF) to prolong the life of the neurons, we also treated the PD rat (treated with 6-OHDA) with induced BMSCs. BMSCs can be cultured in many medias, such as DMEM, a -MEM, DMEM/F12, MesenCult. It is important to compare the result of different lab, whether the grow conditions of BMSCs' subpopulation cells are the same in all the medias. So in the experiment we chose DMEM and MesenCult to observe subpopulation cells' grow condition in the two medias.Methods: BMSCs were separated from rat bone marrow and human bone marrow with Percoll. (1) The primary human BMSCs ( hBMSCs ) were cultured in DMEM and MesenCult respectively, And then observed the grow of BMSCs' subpopulation cells. The cells (from passage 2 or passage 3, P2 or P3) were replated in 96-well plates. And observed the subpopulation cells grow and transformation. (2) When the cells (from P 2 or P 3 ) confluent about 60%-70%, the media were replaced with CSF ,or AA, or BHA /DMSO/ factors, or BHA/DMSO/20% FBS. Observed the morphology of the subpopulation cells of BMSCs at different time. Cells were fixed for immunocytochemistry. Detected the transmitters of catecholamine (CA) in medias by HPLC. (3) PD rats were divided into control group and experiment group at random. Control group was consist of normal control, DMEM media group and BDNF/NT-3 mediagroup. Experiment group was consist of undicuced hBMSCs/DMEM group, induced hBMSCs/DMEM group and induced hBMSCs/ BDNF/NT-3 group. After 4 weeks differentiated, apomorphine-induced rotation was observed. And then the rats were anaesthetized and perfused, fixed, for immunohistochemical.Result: (1) hBMSCs grown faster in MesenCult than in DMEM, and small cells were poor in MesenCult. Small cells were transformable to spindle-shaped cells and flattened cells. Spindle-shaped cells were transformable to flattened cells. (2)After supliment factors (BDNF, NT-3, PDGF. IL-1 , IL-11, LIF) in BHA/DMSO media, the differentiated cells' life could be prolong. The differentiated cells expressed GFAP and NSE. After supliment 20%FBS, the cells morphology transformation were not go on and almost all differentiated cells return to BMSCs. After seven hours induced by 50 u g/mlAA, the cells just transformed. After 3 hours induced with 100-200 u g/mlAA, small cells displayed typical neuronal morphologies, but their apoptotic speed was quick. Flattened cells' transformation was slow. Induced by 500 u g/mlAA, the cells transformation was fastter than the others doses. The differentiated cells expressed GFAP and NF. There was catecholamine in the induced medias. The BMSCs number that had morphology changed in craniopharyngioma patient's CSF more than that in traumatic brain injury patient's CSF. The cells morphologies changed faster hi craniopharyngioma patient's CSF. The differentiated cells expressed NSE and NF, markers of neural stem cells. (3) Four weeks after treated with BMSCs, Apomorphine-induced rotation was observed hi any group. The decreased rotation percentage of DMEM group, uninduced BMSCs/DMEM group, induced BMSCs/DMEM group, BDNF/NT-3 group, induced BMSCs/BDNF/NT-3 group were -6.57%, 100%, 78.70%, 53.63%, 100%, respectively. The others groups were significantly higher than control group, especially uninduced BMSCs group and induced BMSCs/BDNF/NT-3 group(P<0.05). Immunohistochemical staining: TH-positive cells could be observed in the CPU, and TH-positive cells were cluster near MFB hi DMEM group, In BDNF/NT-3 group , TH-positive cells were cluster in inject...