Research On Primary Cultured Olfactory Ensheathing Cells Co-cultured With Heterogeneous Nerve Scaffold

Objective1. Using a modified differential adherence limited joint trypsin digestionmethod neonatal rat olfactory ensheathing cells, and detecting cells secrete nerve growthfactor.2. Primary cultured olfactory ensheathing cells co-cultured with heterologous nervescaffold in vitro observation bracket would affect the growth of cells in vitro environment.Methods Experiment I: Take ten neonatal SD rats (rats aged7d), take the neonatal ratolfactory bulb, using a modified differential adherence joint trypsin digestion time3mincultured cells as a method;After incubation7d detect cells secrete nerve growth factor, adjustthe cell density to the density of1×106/ml, seeded into24-well plates, each density group isdivided into3d,5d group,7d group,9d group, nerve growth factor enzyme linking reactionto detect differences in the cells at different time points in the secretion of growthfactors.Experiment II: Extraction dissimilar nerve scaffold, nerve is taken HE, S-100,Laminin immunohistochemistry; Olfactory ensheathing cells and xenograft nerve scaffoldco-culture in vitro, by the scanning electron microscopic observation the coexistence of cellsand scaffold;Nerve growth factor was detected by enzyme-linked olfactory ensheathing cellsalone (control group), cells co-cultured with the bracket (experimental group) in3d,5d,7d,9d, whether there are differences in the nerve growth factor secreted at different points intime.Results Experiment I：Modified differential adherence joint trypsin digestion method3min limited olfactory ensheathing cells cultured than a simple method for differentialadherent cells cultured at high concentrations,cell concentration can reach70%,；Nerve growth factor content data through statistical analysis of olfactory ensheathing cells3minlimited trypsin digestion secretion gradually decreases with time.After trypsin digestion first3d levels of nerve growth factor secreted the highest (P=0.012, P <0.05). Experiment II:Nomicroscopic heterogeneous nerve scaffold Schwann cells and myelin structure afterextraction, nerve basement membrane, endometrial, beams and adventitia are present;Olfactory ensheathing cells are able to grow on heterogeneous neural attached bracket;experimental group and the control group in3d,5d,7d,9d, differences in the secretion offour different time points on nerve growth factor was not statistically significant (P>0.05)Conclusions1. Olfactory ensheathing cells in co-culture method can achieve higherconcentrations of olfactory ensheathing cells, cell concentration can reach70%, to meet therequirements of post-transplant cells.2. Joint training of olfactory ensheathing cells in cellgrowth to about10d (limited trypsin digestion3d) when the maximum amount of nervegrowth factor secretion.3. After extraction heterogeneous nerve scaffold scaffold as a carrierto meet the requirements.4. Primary cultured olfactory ensheathing cells can catch bracketattached to the nerve growth.5. Olfactory ensheathing cells and scaffold not received afterco-culture influence on nerve growth factor secretion, normal cells can secrete growthfactors, dissimilar bracket does not interfere with the normal growth of nerve cells.