| Objective 1. Identify olfactory ensheathing cells(OEC) from purified primary culture of olfactory mucosa which derived from neonatal SD rat. 2. Investigate the induction of autophagy of olfactory ensheathing cellsin spinal injury under in vitro hypoxic environment, and effect of autophagy on the OEC proliferation. 3. Verify the treatment effect of OEC transplant to hemisection spinal cord injury. 4. Discuss the appropriateness of SPIO in vitro labelling of OEC and its in vitro imaging characteristics.Methods 1. Purified OEC were obtained through digestive enzyme separation process and modified Nash differential adhesion purification process, cell cultures were selected according to the proliferation rate, as per CCK-8 method. Cell properties and purities were identified using immunofluorescence technique. 2. The OEC of neonatal SD rat of hypoxic group were cultivated under hypoxic condition(2% oxygen condition). The OEC of control group were cultivated under regular condition while the 3-MA hypoxic group were cultivated under hypoxic condition(2% oxygen condition) with the presence of autophagocyte inhibitor 3-MA. Cell morphology was observed under phase contrast microscope. Hypoxia-inducible factor 1-alpha(HIF-1α) in all groups were analysed using Western blotting method. The level ofautophagy marker protein LC3 I/II and autophagy marker gene Beclin-1 protein were analysed as well. Control group, Processed group-OECs under normal condition with the use of autophagocyte inhibitor 3-MA\hypoxia group, 3-MA processed hypoxia group, to detect the OECs proliferation ability with CCK-8 method. 3. SD rats were utilised to model left sided hemi section spinal cord injury, OEC were transplanted immediately after the injury to the experimental group while the control group was injected complete cell culture medium and the pure injury group was not given any medication after the injury. The motor function of all groups were evaluated using BBB locomotor scale method at 1st, 2nd, 4th, 6th and 8th week. This would verify the therapeutical effect of OEC transplant to hemi section spinal cord injury for SD rat. 4. Label OEC using polylysine-coated superparamagnetic iron oxide nanoparticles(PLL-SPIO), labelling results were observed using Prussian blue staining and Transmission Electron Microscope, the cell activities and proliferation rate of the labelled OEC were monitored and in vitro MRI images were scanned.Results 1. The OECs that sourced and cultivated from olfactory mucosa, were mostly in spindle shape, classic bipolar and tripolar cell morphology, after the primary culture undergoing separation process and purification process. Cells that are both GFAP and P75 stained positive under laser scanning confocal microscope are OECs. Its purity according to formula could reach 87%. 2. In comparison with the control group, the hypoxic group has higher HIF-1α protein level(P <0.05), significant transformation from LC3 I to LC3 II and higher Beclin-1 protein level(P<0.05) while the 3-MA group has lower proliferation rate(P<0.05). 3. The experimental group, compared with control group and pure injury group, P < 0.05 statistically significant while comparing control group with pure injury group at all time slot P > 0.05 statistically insignificant; As time passes, the BBB score of all groups experience improvement. 4. By successfully utilizing SPIO maker of OECs without affecting its biological feature, it illustrates a better result on MRI invitro(1x106) axis with T2 WI and SWI represent x-and y-axis respectively.Conclusion 1. The hypoxia-induced autophagy improves the proliferation rate of OEC. 2. The transplant of OEC has therapeutic effect on hemisection spinal cord injury. 3. OEC were successfully labelled using SPIO method, without any adverse effect to the basic cell properties and its proliferation rate, and detected by MRI in vitro imaging. This provides the basis to the cell tracking in OEC transplantation to spinal cord in the future. |
