| Back GroundBMSCs are the cells consisted in the bone marrow, which are not belong to blood stem cells (non-adherent cells) but adherent cells because of their property in experiment with plastic plate. They are also named as plastic-adherent cells, clone-forming-unit fibroblast cells or mesenchymal stem cells (MSCs). Recently, some studies indicated that the BMSCs of mammal could differentiate into cells of several tissues, including nerve tissue. BMSCs can be takem out from the auto bone marrow cavity or soft-matter bone easily with less injury and it will not bring on immunoreaction. These cells are non-differentiated and have stronger potential of proliferation. Therefore, we can culture and increase cells number in vitro. Some reporters indicated that BMSCs could be induced into neural stem cells (NSCs), so the BMSCs might be considered as a kind of perfect germ cells.It is focus in the domain of neuroscience recently to transplant NSCs as a treatment way on injury central nerve system (CNS). However, the transplanting of NSCs can not be applied to clinic for that if we gain NSCs from the brain tissue of embryo as we used to do, it will be confronted with some problems such as the quantity of NSCs used in transplant is not enough and the ethnics problem, etc. The discovery of the multi-potential of bone marrow stromal cells and the successful oriented differentiation towards NSCs can offer us a new source of NSCs. The aim of this study is to culture NSCs derived from bone marrow, and then, to transplant the stem cells intobrain to observe these cell's ability of survival and migration. We used macaque as object in this study because we can obtain some parameter from it that is helpful and useful in clinical treatment. The study includes culturing and transplanting of NSCs.Part one: Experimental study on inducement anddifferentiation of neural stem cells in vitroObjective1. To observe the status of the BMSCs which are cultured and induced into NSCs in vitro.2. To explore the feasibility of differentiation from the BMSCs into the NSCs, and to establish the method and condition of BMSCs culture in vitro. And then, to make the NSCs derived from BMSCs as germ cell to be useable in tissue-engineering and clinical medical.Method1. Acquiring bone marrow by puncturing the marrow cavity of SD rats and macaque under aseptic condition respectively. We use density gradient centrifugation to acquire mononulear cells with nucleus and adherence technique to acquire bone marrow stromal cells.2. The cells The density of the cultured cells had been adjust to 106/ml and the cells had been inoculated in Petri dish, which was added in bovine blood serum (1%) , LIF (10ng/ml) and bFGF (10ng/ml), and then cultured in 37℃, 5%CO2 and saturation wetness. Exchange culture medium firstly at 48 hours post inoculation in order to wip off the non-adherent cells and then exchange medium two times per week. In this way, we can obtain relatively purified MSCs and subculture it. In subculture, we add in bovine blood serum and RA (concentration 0.5μg/ml) in order to induce MSCs to NSC.3. Observation of cultured cells: on the CK2 invert phase different microscope (OLYMPAS, JAPAN), we continued observe the cells and take photos. In various stage of culture, we take cells sample to be detected by Immunocytochemical (SABC, first antibodys were Rat anti-Nestin, Rabbitanti NSE and Rat anti-GFAP. Second antibodys were biotinylated Goat anti Mouse IgG/Goat anti Rabbit IgG and streptavidin peroxidase). The sample fixed in 4% polymerisatum firstly, flushed by 0.01M PBS, added first antibody and incubated in 37℃60 min, and then added second antibody incubated in room temperature 10min, thirdly, added streptavidin peroxidase and incubated in room temperature 10min, finally, stained by DAB 5-15min. The positive stained cells are brown. Take picture with OLYMPAS microscope. Result1. We can see division and proliferation in adherent cells in culture, which can form cell clones like islands. When be...
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