Effect Of Cerebrospinal Fluid On The Proliferation And Differentiation Of Neural Stem Cells

CharpterⅠIsolation, Cultivition, ldentification of brain and spinalcord derived neural stem cells from newborn ratObjective: To compare the difference of morphology andproliferation capacity between the neural stem cells(NSCs) from brainand spinal cord of newborn rat, and make a basis for further research onthe transplantation of neural stem cells.Methods: The NSCs of brain and spinal cord in newborn (aged 24hours) Sprague-Dawley(SD) rat were isolated, cultured, expanded andpassed generation in vitro by serum-free culturing technique, and theninduced differentiation in medium supplemented with fetal bovine serumafter removing growth factor. The cells growth, proliferation anddifferentiation were observed under the phase contrast microscope. TheNSCs and the differentiated neural cells were identified byimmunofluorescence technology.Results: The NSCs, derived from brain and spinal cord, have thesame morphology and could self-proliferate. The quantity of neurosphereand the cell population of a neurosphere from brain were more than thatfrom spinal cord. And the proliferation speed of brain derived NSCs isfaster than that of spinal cord derived. Most of the primary and passagedcells from brain and spinal cord showed protein-Nestin positive reaction.Both of the NSCs from brain and spinal cord can differentiate into threemajor cell types of the nervous system and express to specific antigens,such as NSE, GFAP, of neuron and astrocyte.And both cells can keepself-reproduction ability and muliti-directional differentiation potentialafter going down to posterity repeatedly.Conclusion: 1. The NSCs, acquired from the spinal cord and brain ofnewborn rat, have the ability of self reproduction and themulti-differentiation potential.2. The brain-derived neural stem cells have much more number and proliferative ability. Both of brain and spinal cord derived neural stemcells can expand largedly in vitro.CharpterⅡEffect of eerebrospinal fluid on the proliferation anddifferentiation of neural stem cellsObjective: To investigate the effect of cerebrospinal fluid(CSF) onthe survival, proliferation and differentiation of neural stem cells.Methods: NSCs were isolated and cultured from the brain ofnewborn (aged 24 hours) SD rat. After purified through 3 generations, theNSCs were cultured in human hemorrhagic CSF from brain injury patientor CSF from hydrocephalus patient in order to simulate NSCs live indifferent CSF after transplanted in CSF. The cells were divided intoexperimental group(hemorrhagic CSF group) and controlgroup(hydrocephalus CSF group). The growth, proliferation anddifferentiation pattern of NSCs in two groups were observed at differenttimes. The differentiated cells were identified by immunocytochemistryand their proportion were compared.Results: The NSCs can survive both in hemorrhagic CSF andhydrocephalus CSF. Some of the cells grew as floating neurosoheres inCSF, while most of the NSCs attached to the coverslips and differentiatedinto neurons and glia. The attachment and differentiation speed of NSCsin experimental group is faster, the attachment ratio is higher and morecells differentiated completely when compared with that in contral group.And there were NSE~+ neurons and GFAP~+ astrocytes among thedifferentiated cells in two groups. At the first stage of differentiation, thepercentage of NSE positive cells in experimental group is higher than thatin control group, which was confirmed by statistics analysis to besignificant differences(P＜0.05). While at the end of the stage, thepercentage of GFAP positive cells in experimental group is higher and thepercentage of NSE positive cells is lower than that in control group,which was confirmed by statistics analysis to be significantdifferences(P＜0.05).Conclusion: 1. The NSCs can survive,proliferate and differentiateboth in brain injury hemorrhagic CSF and hydrocephalus CSF.2. The attachment and differentiation speed of NSCs in brain injury hemorrhagic CSF is faster, the attachment ratio is higher when comparedwith that in hydrocephalus CSF.3. The brain injury hemorrhagic CSF and the hydrocephalus CSFhave different effect on the differentiation of NSCs. More neural stemcells prone to differentiate into astrocyte in brain injury hemorrhagic CSF,while more neurons in hydrocephalus CSF.