The Study Of Co-culture Of Rat Olfactory Ensheathing Cells (OECs) And Human Umbilical Cord Mesenchymal Stem Cells(HUC-MSCs)

ObjectiveTo observe the influence of co-culture of rat olfactory ensheathing cells (OECs)and human umbilical cord mesenchymal stem cells (HUC-MSCs) in vitro environ-ment on the survival, the proliferation and the differentiation of neural cells; Todescribe the expression of nest protein (nestin) induced by HUC-MSCs, the myelinbasic protein(MBP) and high molecular weight neurofilament(NF-H), and to explorethe mechanism in the process of the neural differentiation of HUC-MSCs promotedby OECs.Methods1. The OECs of healthy wistar rats (3months old) were cultured and purifiedunder sterile condition. By the method of Ara-c suppression combined with differen-tial adhesion and OECs were identified by immunohistochemistry. The umbilicalcords of healthy fetus with uterogestation were selected under sterile condition. TheHUC-MSCs were cultured by adherent method. The HUC-MSCs of P5wereidentified with flow cytometry.2. Firstly, the experiment were classified into3groups: the co-cultured two cellswas experimental group, the isolated cultured two cells was control group, and blankcontrol group(with DMEM only). All the groups were cultured under5%CO2，37℃c ondition.(1) Observe the growth, rejection reaction of the cells by the phase-contrastmicroscopy on the first day，3rd day,7th day and14th day.(2) Add with CCK-8, the culturing went on for1h. Secondly, the proliferation of all the groups was texted by MTT. Finally, the data was analyzed by SPSS16.0.(3) Test the expression of nestin, MBP, and NF-H in HUC-MSCs byimmunofluorescence technique at3different time points, and then the percentage ofpositive cells was calculated. The immunofluorescence PCR was performed to assessthe expression of nestin, MBP, and NF-H in HUC-MSCs. Finally, the data wasanalyzed by SPSS16.0.Result1. After rat olfactory ensheathing cells (OECs) and human umbilical cordmesenchymal stem cells (HUC-MSCs) were primary cultured and purified, we couldget the purity of more than90%of the cells.2. Cultured cells grew well, had a good cell proliferation, two kinds of mixedlymphocyte culture showed no rejection.3. MTT showed that OD of the mixed culture group was significantly higher thanthat of single cell group’ OD, the difference was statistically significant (P<0.01)4. The immunofluorescence result showed no expression of nestin, MBP, andNF-H in Group C; however, Group A and B were immunopositive for nestin, MBP,and NF-H, and the percentage of immunopositive cells in Group A is higher thanGroup B.5. The RT-PCR results showed that nestin, MBP, and NF-H expressed on mRNAlevel in group A and B, and that was significantly higher in group A than in group B.Conclusion1. Between the rat olfactory ensheathing cells and human umbilical cordmesenchymal stem cells after mixed culture, cell growth condition was good, norejection reaction between the cells.2. The total number of mixed group of cells was much more than that ofsingle cells, the growth and proliferation of cells was signifycantly better than that ofsingle cells.3. The OECs and the HUC-MSCs can be co-cultured in vitro. The proliferation ofthe two cells was promoted after co-cultured. The OECs can also promote thedifferentiation of HUC-MSCs to neural cells, and some of the HUC-MSCs expressmarkers of neuroepithelial stem cells, neuron and oligodendrocyte. The expression ofnestin, MBP, and NF-H showed a characteristic of time dependency.