In Vitro Study On Biological Characteristics Of Neural Stem Cells And Oecs Derived From Olfactory Mucosa

Objective:To establish the method for culturing neural stem cells (NSCs) from olfactory mucosa, optimize the conditions of neural stem cells subculture, and induce neural stem cells to differentiate into dopaminergic neurons, so that fundamental research data can be provided for stem cell therapy for Parkinson's disease (PD). To find the method for culturing and purifying olfactory ensheathing cells (OECs) derived from olfactory mucosa, to investigate the neurotrophic effects of OECs, so as to discuss the possibilities of using OECs derived from olfactory mucosa as chaperone cells during cell therapy of PD.Methods:1. The nasal olfactory mucosa was removed alive from the adult Sprague-Dawley (SD) rats, made into cell suspension, and cultured in DMEM/F12 medium added with 10% fetal bovine serum for 3 days, and then cultured in medium added with growth factors specific for NSCs. The adhesion, proliferation and differentiation of primary cultured cells were dynamically observed. Immunofluorescence staining was applied to detect the expression of NSCs specific molecular markers on these cells.2. A simple anti-adhesion method using 2% agarose gel was applied to subculture neurospheres derived from olfactory mucosa. The biological characteristics of the passaged neurospheres were studied using immuno-fluorescence staining and western blotting.3. A combination of three factors including neural growth factors, ATRA and Shh was utilized to induce neurospheres to differentiate into cells synthesizing tyrosine hydroxylase (TH). Immunofluorescence staining and western blotting were applied to confirm that these cells were dopaminergic neurons expressing neurofilament and TH.4. Dissected olfactory mucosa from adult SD rats, and then cultured the dispersed cells from them with complete medium in vitro. A method based on different adhesion time was applied to purify OECs for three times, and then immunofluorescence staining was used to determine the OECs.5. Compared the different levels of neurotrophins, neuropeptide Y and VEGF in primary OECs and OECs passaged for 10 times by immunofluorescence staining and western blotting.Results: 1. The flatten cells adhered first followed by globe like cells after the mixed cells from olfactory mucosa were seeded into flasks. After complete medium was changed into NSCs specific medium 3 days later, a large quantity of globe like cells with a tendency to aggregate could be observed. Lots of nest-like semi-adherent neurospheres could be spotted after 7 days. The result of immunofluorescence staining showed that adherent globe like cells expressed Nestin and CD 133 positively, with Nestin locating in cytoplasm and CD 133 locating on cell membrane. The semi-adherent neurospheres were also Nestin and CD 133 immunoreactive.2. The passaged NSCs could proliferate quickly as neurospheres cultured with 2% agarose gel anti-adherent method. The subcultured neurospheres had regular morphology, same size and clear boundary. The neurospheres in suspension passaged for 4 times were Nestin and CD 133 immunoreactive after adhesion for 6 hours as shown by immunofluorescence staining and western blotting.3. Treatment of adherent neural stem cells with a cocktail of 3 different cytokines could lead some of them to project long and thin processes which were netlike connected later. These cells with lots of processes were NF and TH immunoreactive as shown by immunofluorescence staining and western blotting.4. Spindle shaped bipolar cells could be obtained by expanding OECs which had been undergone a purification process based on different adhesion time for 3 times. These bipolar cells had similar shapes and arrayed regularly as a flock. Both of the primary and passaged OECs were GFAP, S-100 and p75NTR immunoreactive alike as shown by immunofluorescence staining.5. Both of the primary and passaged OECs could synthesize 3 kinds of neutrophins, neuropeptide Y and VEGF with the former had stronger capacity than the latter.Conclusions:1. We successfully obtained semi-adherent neurospheres which were Nestin and CD 133 immunoreactive by dissecting olfactory mucosa from alive rats. These neurospheres could proliferate continuously by applying a specific expansion method with agarose playing a pivotal role.2. The olfactory mucosa derived neurospheres could be induced to differentiate into TH positive neurons. So neural stem cells derived from olfactory mucosa could become dopaminergic neurons.3. We got a cell society by dissecting olfactory mucosa and then cultured it with complete medium followed by purifying the OECs from this society with a method based on different adhesion time. The purified OECs we obtained could synthesize 3 kinds of neurotrophins, NPY and VEGF.4. In general, We successfully obtained neural stem cells and olfactory ensheathing cells from olfactory mucosa, and got dopaminergic neurons from these neural stem cells. These results shed some light on finding a new source of seed cells for cell therapy of PD.