Study On Co-culturing Neural Stem Cells With Olfactorybulb Ensheathing Cells From Olfactory Mucosa And Transplanting To Cure Fimbria-fornix Transection Rats

Objective:Firstly, the aim is to compare the proliferation, differentiation and biology property ofolfactory ensheathing cells derived from olfactory mucosa and bulb. Secondly, to observethe effect of proliferation and differentiation of NSCs by co-culturing NSCs with olfactorymucosa OECs. Thirdly, to observe survival and differentiation of NSCs after transplantingNSCs and olfactory mucosa OECs into fimbria-fornix transection rats. Based on these,applying NSCs and OECs to treat diseases of nervous system.Methods:1. OECs derived from mucosa and olfactory bulb were cultured in vitro by differingrates of attachment. The proliferation of OECs were observed and recorded by theinverted microscope everyday.2. The configuration of OM and OB OECs were observed by scanning electronmicroscope (SEM).3. The content of NGF and BDNF in OM and OB OECs culture supernatant wasdetected by ELISA.4. OM and OB OECs were marked with the immunofluorescence antibody ofp75NGFR and S-100to observe their proliferation and differentiation.5. Co-culturing NSCs and olfactory mucosa OECs in direct contact group is directlymixing these two kinds cells into24wells plate. In indirect contact group, using transwellplate, OECs were planted on superstratum and NSCs on the bottom. The control group isonly planted NSCs. Compare the neuron differentiation of NSCs.6. NSCs in NSCs group, NSCs at the bottom of transwell plate in transwell group,OECs and NSCs in the direct contacting group were transplanted into the bilateralhippocampi dentate gyrus of fimbria-fornix transection rats. The control group only inject normal saline (NS). After2w,4w,6w and8w, rats were executed behavior tests includingdark to avoid detection test and platform test. Then perfused by rat heart, took out and dothe frozen section of rat brain. The double label of BrdU and MAP-2immunofluorescenceantibody was used to detect survival and differentiation of NSCs after transplanting.Results:1. There is no distinct difference on the appearance, the number and the volume ofOECs sphere between OM and OB OECs at the same culture period.2. SEM showed that most of OM OECs are bipolar cells. OB OECs have three kindsof shapes. They are bipolar rcells, tripolar cells and round cells. Most of them are bipolarcells.3. Results of Elisa showed that there were NGF and BDNF in OM and OB OECsculture supernatant. The content heats up gradually with the time. There is no distinctdifference on the content of NGF and BDNF in OM and OB OECs culture supernatant atthe same culture period.4. S-100and p75NGFR immunofluorescence results showed that there were manyp75NGFR and S-100double labeled positive cells in OM and OB OECs. Most of OMOECs are bipolar cells. Cell body of OM OECs is slightness and fusiformis. OB OECshave many kinds of shapes. They are bipolar rcells, tripolar cells and round cells. Cell bodyof OB OECs is long fusiformis, triangle and round. There is no distinct difference on thepurifying ratio, the soma area and perimeter of OM and OB OECs.5. Many MAP-2positive cells were seen in the direct contact group and the transwellgroup. These cells have is big cell body, long neurite and abundant ramification. The cellbody of MAP-2positive cells in the direct contact group is bigger than other groups. Theratio of MAP-2positive cells and cell perimeter in the direct contact group and thetranswell group are greater than the control group. To compare the direct contact group andthe transwell group, cell perimeter in the direct contact group is greater than the transwellgroup. But there is no obvious difference the ratio of MAP-2positive cells between thedirect contact group and the transwell group.6. The results of behavior test after cell transplantation showed that there was obviousdifference on the frequency of exploration and the time of stagnation of the direct contactgroup comparing with other three groups. To compare with NSCs group and transwellgroup, the direct contact group differs greatly in the frequency of exploration. The rate of active avoidance and the total rate of avoidance of three cell transplantation groups arediffer from the control group. The results of pairwise comparison showed that the directcontact group had obvious difference compared with orther groups. The results of thedouble label of BrdU and MAP-2immunofluorescence antibody in three celltransplantation groups showed that there were BrdU and MAP-2double positive cells intransplantation area of brain slice of three cell transplantation groups. The number of BrdUand MAP-2double positive cells in the direct contact group and the transwell group aremore than the NSCs group. Cell body of BrdU and MAP-2double positive cells in thedirect contact group is bigger than transwell group and NSCs group.Conclusions:1. OM OECs, the same as OB OECs, can be cultured and proliferated in vitro. Mostof OM OECs are bipolar cells. OB OECs have many kinds of shapes. They are bipolarrcells, tripolar cells and round cells. OM OECs, the same as OB OECs, can excrete NGFand BDNF. There is no distinct difference on the shape of differentiation cells between OMOECs and OB OECs.2. The ratio of differentiation neuron from NSCs is obviously increased when NSCsdirect or indirect contact co-cultured with OECs. OECs can promote the growth andmaturation of neuron when NSCs direct contact co-cultured with OECs.3. The behavior of fimbria-fornix transection rats can be improved after celltransplantation in NSCs group, transwell group and direct contact group. OECs canpromote the survival and differentiation of neuron when NSCs direct contact with OECs invivo.