| Objective: The in vitro culture method of nerve tissue is a valuable research tool of modern neuroscience. Dorsal root ganglion neurons (DRGn) are the sensory neurons of PNS(peripheral nerve system), DRGn in vitro model has been applied widely to research the Neuronoscience including the neurite guidence, the development and plasticity of synapse, myelination in the PNS and CNS (central nerve system), the effect of neurotrophic factor and the distribution of their receptors,the mechanism of aging in nerve cells, gene therapy and Tissue Engineering , in addition to provide a new means for studing heritable, acquired conditions. Guillain-Barre syndrom (GBS) belongs to diseases of the peripheral nerves and includes many clininic subtype. At present it has been considered that GBS is autoimmune neurological disease mediated by humoral and cellular immunity. However its pathogenesis is unknown completely. This experiment established DRGn in vitro model to produce a brand new means for furthering the study of GBS. This paper presented DRGn isolated and purified in vitro culture systems from embryonic rat, and investigatedthe biological property of cultured DRGn from cell morphology at the level of the light microscope. Methods: (1)The mixed DRGn culture system was established by applying the monoplayer primary dissociated culture method of nerve tissue .DRGs were removed asepticly from 15-day Sprague-Dawley (SD) embryonic rat., and digested with trypsin for 20 minutes, then triturated to produce single cell suspension. The individual cells at a density of 106/ml were then plated in tissue culture plates precoated by PLL (poly-L-lysin)and LN (laminin), then cultured in NB1 media supplemented with GDNF (glial cell line-derived neurotrophic factor ) at CO2 incubator. (2) Purified DRGn culture system was established by differential adhesion. Firstly single cell suspension that was produced by trypsin was plated on uncoated 35mm culture dish and incubated for 50 minutes at CO2 incubator. Unadhered cells were harvested and plated in tissue culture plates precoated by PLL and LN, and then cultured in NB1 media. (3) The growth regularity of DRGn in vitro was observed under phase-contrast microscope at different time , which included the survival of neuron, the change of soma and the outgrowth of the neurite. DRGn were also observed using Nissle staining , and identified using neuronal specific enolase (NSE) antibody immunohistochemical staining. The numbers of NSE-positive neurons were counted in mixed and purified culture system , and the percentage of NSE-positive neuronswere evaluated to show the purified rate. Results: Cultured DRGn were capable of suriving under the suitable conditions in vitro. After 12-18 culture hours the cells had adhered to the culture plate with a few neurites .The increasing and enlonging neurite reaggregated to dense network between cells. During the whole culture course, nerve cells altered their shape from a spherical to a spindle,triangle and polyangle-like forms. This change was not associated with the change in cell size. The neurons could matured gradually with the culture, which showed that the rate of nuclei and cytoplasm was increasing. Some neurons could die at different culture periods. Mixed culture system containing DRGn, Schwann cells and fibroblasts was be able to maintained 3-4 weeks, While purified culture system which mainly contained neurons could survived 2 weeks. Granule- and lump-shape Nissle bodies which were identified by Nissle staining distributed in cytoplasm of cultured DRGn, thus meaned that the cultured DRGn survived healthily. DRGn were identified using NSE immunohistochemistry. NSE-positive cells were observed in mixed and purified culture system, and the purification rate was calculated and arrived at 31.12±3.2% ; 91.23±2.9% respectively. There was marked statistics difference between the two culture systems. Thus high purification rate DRGn could be harvested in culture system by differential adhesion. Conclusions: The experiment establ... |
PDF Full Text Request