| Objectiveneural stem cells are the special cell group in the early developing stage of nerve systems. Due to their abilities to proliferate and differentiate, they bring hoperess to the treatment of nerve system impairment and degeneration lesion. To provide support, we promoted neuronal differentiation in neural stem cells and the mechanisms has been in vestigated.our experiment is divided into three steps. Step 1: separate and culture the spinal cord derived neural stem cells of the new-born rats. Add NCR, IGF-1 and insulin to make them differentiate into cholinergic neurons.Step two: observing the change of expression of the cell skeleton protein P-tubulin during the spinal cord neural stem cells' differentiating into neurons. During the development of the growth and movement of the cells, the issue of axis-cylinders and dendrition, the branches which carry the big molecular substance and cell speculum to their processus. And axis-cylinder's building synapse connection with other nerve cell is associated with the cell framework. As well know, the richest framework protein in the neuron is -tubulin. As a result, the changes of -tubulin can take effect on the differentiation of the neural stem cells. Andp-catenin can better reveal the Processus growth mechanism during the development of the neurons.Step three: observe the change of GAP-43 during Processus growth cone during the neural stem cells ' differentiating into neurons and relationship betweenp-tubulin and GAP-43 during the growth of the processus with Confocal Laser Scanning Microscope, which can detect and identify the distribution and expression of -tubulin and GAP-43 in the neur and hence analysis their three-dimensional structure and further reveal their functions in the growth of the neur-processus.Experimental Methods1. separate and culture the spinal cord derived neural stem cells of newborn ratsWistar rats bom within 24h, take out of the spinal cord aseptically. Cut up, digestion, centrifugation. it and add DMEM/F12 nutrien without serum of B27, bFGF and EGF and cultivate it in the incubator. Collect nerve sphere after seven days and suspend into mono-cell suspension and grow it in the 96-aper-tureed cultivating board. Passage in the same way for four times. Perform the immunocytochemistry of Nestin.2. Commited differentiation and Identification of spinal cord derived-neural stem cellsThe pro-generation and secondary generation cells which were Centrifugated and suspend into single cells, and grow them in the 6-apertured petri dish with cover glass and ACLAR. The cultivation aperture is divided into experiment group and controlled group: add DMEM/12 culture medium with 10% fetus bovine blood serum in the controlled group; add DMEM/12 culture medium with 10% fetus bovine blood serum, NGF, IGF-1 and insulin in expriment. Evaluate the neuron by immunocytochemistry staining and argentation staining methods. Superscript 10 cover glass immuned and stained by NSE from controlled group and experiment group respectively in the seventh day of the differentiation. Count the cells after restained by sap pan lignins. Superscript 10 sight from each cover glass under the multi-timed telescope, count the total number of the cells and NSE positive cells, add up the neuronal ratio, compare the neuronal ratio of the controlled group with that of the experiment group.3. Perform the immunocytochemistry of SABC method and the immu-moflurescence method during the process of the differentiation of the spinal cord derived nerve stem cellOn first fourth and seventh days after the differentiation , superscript the cover glass with cells were taken. Fixed the cover glass with 4% fonnaldehydumpolymerisation, perform theACHE, -tubulin, p-catenin, GAP-43 immuncyto-chemistry with SABC method and immumofluorescence method, meanwhile immunity electron microscope stain them with ACLAR coats to detect the relation between chron-and-nul expression of the ACHE,p-tubulin,p-catenin,GAP-43 during the oriented differentiation of the spinal co...
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