Purification Identification And Characteristics Of Neural Stem Cells In Fetal Rat's Brain

Just As the stem cell study has increasingly maintained much attention, research of neural stem cell is also a focus in the field of neural development and clinical neuroscience. Many groups are interested in the development and regeneration of fetal and adult brain correlated with the concept of neural stem cell. People also hope to transplant neural stem cells into injuried or degenerated brains to rescue the function even to treat some malignant tumor in brain. Progressively all the efforts have achieved lots of resultsThe concept of neural stem cell arises from the field of blood stem cell research and confirms the suspect of regeneration of neural tissue in central nerve system. At the same time, many results about the biological features of neural stem cell in vivo and in vitro induce the hope of future applying of neural stem cell on clinical research. In the theses we discuss the in vitro characterization and purification as well as long term culture of neural stem cells. We will also show some dates of transplantation of purified neural stem cells into adult rat brain.Brain tissues of fetal rats for 16-18 days were homogenated and centrifuged so that the membrane protein separated. SDS-PAGE electrophoresis and transporting the protein to NC membrane provide the target protein lane followed with western blot. The resultsuggests that there is nestin protein in the membrane protein sample, so we got the evidence of nestin distribution in the membrane. To determine the specific antigen of neural stem cell with flow cytometry, fetal and adult rat brain were microsurgically obtained and fixed with monoclonal antibody of nestin, the second antibody of Ig-PE were determined with flow cytometry. The experiments demonstrated (1)nestin positive cells lies in the cortex and subcortex zone of fetal rat brain, suggesting that nestin positive neural stem cell may participates the forming of the central nerves system, (2)The percentage of nestin positive cells in the subcortex zone of fetal rat brain reached 35.4%,leading to the recognition of the subventricular zone or subependymal zone being the central of neural regeneration, (3)few nestin positive cells can be detected in the cortex of adult rat brain but still 5.2% cells in the subventricular zone are nestin positive. Determination of the nestin protein of neural stem cell with flow cytometry is a reliable method.We also try the sorting of high purity neural stem cells from fetal rat brain cells. MACS system is designed to separate or delete cells by antigen-antibody reaction on the surface of target cells. Fetal rat brain cell suspensions were incubated with monoclonal antibody of nestin, and the labeled cells were separated from the suspension in the magnetic field by immunobeads coated with the second antibody. The sorted cell's purity was determined with flow cytometry. The sorted neural stem cell's purity was determined as 93.0%-99.7%. Magnetic cell sorting system can effectively separate neural stem cell from brain cell suspension. A few groups have attempted to establish a neural stem cell line. Culture of cell line in vitro provides the biological features of neural stem cell directly. However for the reason of methods of transferring virus or oncogene into neural progenitor cells may resulted the change of cell growth we try to culture nature neural stem cells sorted by MACS system. During the Long term serum free culture of purified neural stem cells with the medium of DMEM/F12 and the N2 supplyment, EGF shows the ability of enhancing proliferation of neural stem cell. During the culture period, the cell purity is higher than 75%. The purified neural stem cell transplanted into rat brain survived 4 weeks long and differentiated into newborn neurons and glials.In this theses we reported part of work of our group and some conclusions can be listed as followed:(1)other than being part of cytoskeleton, nestin distributed in the neural stem cell membraneï¼›(2) Determination of the nestin protein of neural stem cell with...