| Spinal cord injury is a kind of extremely severe damage It's results in the high disability rate and mortality. The traditional therapy of spinal cord injury usually included spine fracture -dislocation stabilization, relieve spinal cord compression, symptom therapy and rehabilitative treatment. On recent, following development of study of neuro-pathophysiology and neuro-auxology , the transplantation of nervous tissue and cells as a new therapy method is gradually being applicated to the treatment of spinal cord injury, and some satificated were found duringthe therapy result.The bone marrow stromal cells (BMSCs) are a part of bone marrow remove hemopoietic stem cells. It was confirmated recently that the bone marrow stromal cells in under definite culture condition can differentiate into a variety of cell, for instance mesenchymal cell, fibroblast, osteoblast, chondrocyte, adipocyte muscle Cells, endothelial cell and neuron, glial cell and so on, passing clone proliferation. Detected of bone marrow stromal cell pluripotency and succeed of toward nerve cell directional differentiation, increase new pathway for neural stem cells source. Also provide a new idea for solve "self- neural stem cells" transplantation project cell quantity short of problem.Through establish vitro and vivo experiment, to study the bone marrow stromal cellss amplification, orient differentiation and survival in the injuried spine cord to comprehend the biology character of the bone marrow stromalcells, explore the feasibility and the validity of isolating neural stem cellsderived bone marrow, in order to treat disease of the central nervous systemby transplantation of auto- NSCs in future clinic application.Part one: Experimental study on inducement and differentiation ofneural stem cells derived BMSCs of rabbit in vitro1 Objective:(1) 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. 2 Method:(1) Acquiring bone marrow by puncturing the marrow cavity of rabbit 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 Fetal Calf serium (1%) , LIF (10ng/ml) and bFGF (10ng/ml), and then cultured in 37C, 5%C02 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 BMSCs and subculture it. In subculture, we add in bovine blood serum and RA (concentration 0.5 u g/ml) in order to induce BMSCs 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 cell' s samples to be detected by Immunocytochemical (SABC, first antibodys were Rat anti-Nestin, Rat anti NSE. Second antibodys were biotinylated Goat anti Mouse IgG/Goat antiRabbit IgG and streptavidin peroxidase). The sample fixed in 4% polymerisatum firstly, flushed by 0.01M PBS, added first antibody and incubated in 37C60 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. 3 Result:(1) We can see division and proliferation in adherent cells in culture, which can form cell clones like islands. When being continuous cultured, and given appropriate differentiated condition, these cells can differentiate into poly-f... |
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